TW202221119A - Dna-binding domain transactivators and uses thereof - Google Patents

Abstract

In some aspects, the disclosure relates to recombinant adeno-associated viruses (rAAVs) comprising a nucleic acid encoding a fusion protein comprising a DNA-binding domain and a transcriptional regulator domain and methods of using the same. In some embodiments, expression of the fusion protein results in modified expression of a target gene in a cell.

Description

DNA binding domain transactivators and uses thereof

The regulation of target gene expression has become one of the main areas of biomedical research. Upregulation of gene expression corrects haploinsufficiency caused by reduced gene expression. Haploinsufficiency usually results when one or more loss-of-function mutations are present in at least one copy of a gene. AAV-based gene enhancement methods for the treatment of diseases associated with haploinsufficiency are hampered by the packaging capabilities of traditional rAAV vectors.

Aspects of the invention relate to isolated nucleic acids and recombinant AAV vectors for gene delivery. The present invention is based in part on compositions (eg, rAAV vectors and rAAV) and methods for modulating the expression of a target gene, wherein the target gene is haploinsufficient, such as SCN1A. In some embodiments, the present invention provides fusion proteins comprising a DNA binding domain, such as a Cys2-His2 zinc finger protein (ZFP), and a transcriptional regulatory domain. In some embodiments, the compositions described by the present invention include fusion proteins comprising a DNA binding domain (eg, a ZFP, a transcription activator-like effector (TALE) domain, etc.) fused to a transcriptional regulatory domain. In some embodiments, the fusion proteins described by the present invention increase the expression of a target gene (eg, SCN1A) and are therefore useful in the treatment of diseases characterized by defective expression of the target gene in cells or individuals compared to normal cells or individuals (eg, diseases associated with target gene haploinsufficiency).

Accordingly, in some aspects, the invention provides an isolated nucleic acid comprising a transgene configured to express at least one DNA binding domain fused to at least one transcriptional regulatory domain, wherein the DNA binding domain binds to A target gene or a regulatory region (eg, enhancer sequence, promoter sequence, repressor sequence, etc.) of a target gene (eg, in an individual or cell), wherein the target gene encodes a potential-gated sodium channel (eg, _Nav 1.1) . In some embodiments, the target gene is the SCN1A gene. In some embodiments, the transgenic line is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). In some embodiments, the at least one DNA binding domain binds to a target gene (eg, in an individual or cell) and the transcriptional regulatory domain modifies (eg, upregulates) target gene expression.

In some aspects, the invention provides a recombinant AAV (rAAV) comprising a nucleic acid comprising a transgene encoding at least one DNA binding domain fused to at least one transcriptional regulatory domain, wherein the DNA binding domain binds to a target gene or a target gene A regulatory region (eg, in an individual or cell), wherein the target gene encodes a potential-gated sodium channel (eg, Nav1.1), and at least one capsid protein. In some embodiments, the target gene is the SCN1A gene. In some embodiments, the transgenic line is flanked by AAV inverted terminal repeats (ITRs).

In some embodiments, at least one DNA binding domain binds to a target gene (eg, in an individual or cell) and the transcriptional regulatory domain modifies (eg, upregulates) the expression of the target gene in the individual.

In some embodiments, at least one DNA binding domain binds to a non-translated region of a target gene. In some embodiments, the DNA binding domain binds to the regulatory region of the target gene, selectively enhancing subsequences, promoter sequences and/or repressor sequences.

In some embodiments, the DNA binding domain binds between 2 and 2000 bp upstream or between 2 and 2000 bp downstream of a regulatory region (eg, enhancer sequence, promoter sequence, repressor sequence, etc.) of the target gene.

In some embodiments, at least one DNA binding domain encodes a zinc finger protein (ZFP), a transcriptional activator-like effector (TALE), a dCas protein (eg, dCas9 or dCas12a), and/or a homeodomain. In some embodiments, at least one DNA binding domain binds to the nucleic acid sequence set forth in any one of SEQ ID NOs: 5-7. In some embodiments, at least one DNA binding domain binds to at least 2 (eg, at least 3, 4, 5, 6, 7, 8, 9, 10 or more) of the nucleic acid sequences set forth in SEQ ID NO: 3 consecutive nucleotides. In some embodiments, the at least one DNA binding domain comprises a helix-recognizing zinc finger protein encoded by a nucleic acid having the sequence set forth in any of SEQ ID NOs: 11-16, 23-28, or 35-40. In some embodiments, at least one DNA binding domain is a zinc finger protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 17-22, 29-34, or 41-46.

In some embodiments, the at least one DNA binding domain is a zinc finger protein comprising a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 11, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 12, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 12, The recognition helix encoded by the nucleic acid comprising SEQ ID NO: 13, the recognition helix encoded by the nucleic acid comprising SEQ ID NO: 14, the recognition helix encoded by the nucleic acid comprising SEQ ID NO: 15 and/or the nucleic acid comprising SEQ ID NO: 16 Coded recognition helix. In some embodiments, the at least one DNA binding domain is a zinc finger protein comprising the amino acid sequence of SEQ ID NO:57. In some embodiments, the ZFP that binds to the SCN1A gene comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least the amino acid sequence of SEQ ID NO: 57 90%, at least 95%, at least 97% or at least 99% sequence identity.

In some embodiments, at least one DNA binding domain is a zinc finger protein comprising a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 23, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 24, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 24, The recognition helix encoded by the nucleic acid comprising SEQ ID NO:25, the recognition helix encoded by the nucleic acid comprising SEQ ID NO:26, the recognition helix encoded by the nucleic acid comprising SEQ ID NO:27 and/or the nucleic acid comprising SEQ ID NO:28 Coded recognition helix. In some embodiments, the at least one DNA binding domain is a zinc finger protein comprising the amino acid sequence of SEQ ID NO:59. In some embodiments, the ZFP that binds to the SCN1A gene comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least the amino acid sequence of SEQ ID NO: 59 90%, at least 95%, at least 97% or at least 99% sequence identity.

In some embodiments, at least one DNA binding domain is a zinc finger protein comprising a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 35, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 36, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 36, The recognition helix encoded by the nucleic acid comprising SEQ ID NO:37, the recognition helix encoded by the nucleic acid comprising SEQ ID NO:38, the recognition helix encoded by the nucleic acid comprising SEQ ID NO:39 and/or the nucleic acid comprising SEQ ID NO:40 Coded recognition helix. In some embodiments, the at least one DNA binding domain is a zinc finger protein comprising the amino acid sequence of SEQ ID NO:61. In some embodiments, the ZFP that binds to the SCN1A gene comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least the amino acid sequence of SEQ ID NO: 61 90%, at least 95%, at least 97% or at least 99% sequence identity.

In some embodiments, at least one DNA binding domain is a zinc finger protein comprising a recognition helix comprising the amino acid sequence of SEQ ID NO: 17, a recognition helix comprising the amino acid sequence of SEQ ID NO: 18, comprising The recognition helix of the amino acid sequence of SEQ ID NO: 19, the recognition helix comprising the amino acid sequence of SEQ ID NO: 20, the recognition helix comprising the amino acid sequence of SEQ ID NO: 21 and/or the recognition helix comprising the amino acid sequence of SEQ ID NO: 21 : The recognition helix of the amino acid sequence of 22.

In some embodiments, at least one DNA binding domain is a zinc finger protein comprising a recognition helix comprising SEQ ID NO: 29, a recognition helix comprising SEQ ID NO: 30, a recognition helix comprising SEQ ID NO: 31, comprising The recognition helix of SEQ ID NO:32, the recognition helix comprising SEQ ID NO:33 and/or the recognition helix comprising SEQ ID NO:34.

In some embodiments, at least one DNA binding domain is a zinc finger protein comprising a recognition helix comprising SEQ ID NO: 41, a recognition helix comprising SEQ ID NO: 42, a recognition helix comprising SEQ ID NO: 43, comprising The recognition helix of SEQ ID NO:44, the recognition helix comprising SEQ ID NO:45 and/or the recognition helix comprising SEQ ID NO:46.

In some embodiments, at least one DNA binding domain is a catalytically inactive CRISPR-associated protein (Cas protein). In some embodiments, the catalytically inactive Cas protein (or "dead Cas protein") is a dCas9 or dCas12 protein. In some embodiments, the nucleic acid or rAAV additionally comprises at least one guide nucleic acid (eg, guide RNA or gRNA). In some embodiments, the guide nucleic acid comprises a spacer sequence targeting SCN1A. In some embodiments, the guide nucleic acid comprises a spacer sequence having the nucleotide sequence of any of SEQ ID NOs: 85, 86, 89, 90, 93, or 94. In some embodiments, the guide nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 83-94. In some embodiments, the guide nucleic acid is encoded by the nucleic acid sequence set forth in any one of SEQ ID NOs: 83-94.

In some embodiments, at least one transcriptional regulatory domain comprises the following transactivator domains: VP16 domain, VP64 domain, Rta domain, p65 domain, Hsf1 domain, TCF4 domain, MEF2A domain, MEF2C domain, MEF2D domain, Sp1 gluten rich domain Amino acid domain, p53 domain, E2F1 domain, MyoD domain, MAPK7 domain, NF1B proline-rich domain, RelA domain, or any combination thereof, such as a VPR domain (VP64+p65+Rtal domain). In some embodiments, at least one transcriptional regulatory domain is encoded by a nucleic acid sequence as set forth in SEQ ID NO:47. In some embodiments, the at least one transactivator domain comprises the amino acid sequence set forth in SEQ ID NO: 48 or any one of 122-134.

In some embodiments, the nucleic acids described herein additionally comprise a nuclear localization sequence (eg, a sequence comprising any one of SEQ ID NOs: 135-140).

In some embodiments, the ITR flanking the transgene comprises an ITR selected from the group consisting of: AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV8 ITR, AAVrh8 ITR, AAV9 ITR, AAV10 ITR or AAVrh10 ITR. In some embodiments, the ITR is ΔTR or mTR.

In some embodiments, the transgenic line of the isolated nucleic acid is operably linked to a promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the tissue-specific promoter is a neuronal promoter, such as SST, NPY, phosphate-activated glutaminase (PAG), vesicular glutamate transporter-1 (VGLUT1), glutamate depleted Carboxylases 65 and 57 (GAD65, GAD67), Synaptophysin I, a-CamKII, Dock10, Prox1, Microalbumin (PV), Somatostatin (SST), Cholecystokinin (CCK), Calbindin (CR) ) or neuropeptide Y (NPY).

In some embodiments, the DNA binding domain of the transgene is fused to the transcriptional regulatory domain via a linker domain. In some embodiments, the linker domain is a flexible linker, such as a glycine-rich linker or a glycine-serine linker or a cleavable linker such as a photocleavable linker or an enzymatically (eg , protease) cleavage linker).

In some embodiments, the isolated nucleic acid comprises a transgene encoding multiple DNA binding domains (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 DNA binding domains). In some embodiments, the isolated nucleic acid comprises a transgene encoding multiple transcriptional regulatory domains (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 transcriptional regulatory domains).

In some embodiments, the isolated nucleic acid or rAAV line is expressed in cells or individuals characterized by abnormal expression or haploinsufficiency (eg, increased or decreased expression) of the target gene relative to normal cells or individuals. In some embodiments, the isolated nucleic acid or rAAV line is expressed in cells or individuals characterized by defective (eg, reduced) expression of the target gene relative to normal cells or individuals. In some embodiments, the target gene of the isolated nucleic acid or rAAV is SCN1A.

In some embodiments, the AAV capsid serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, or AAV.PHPB.

In some aspects, the present invention provides methods of modulating the expression of target genes. In some embodiments, the methods of the invention comprise administering an isolated nucleic acid or rAAV as described herein to a cell or individual expressing a target gene, wherein the target gene is haploinsufficient (eg, SCN1A haploinsufficiency). For example, in some embodiments, the expression of a target gene (such as SCN1A) in a cell or individual is deficient (eg, reduced) relative to the expression of the target gene in a normal cell or individual. In some embodiments, the isolated nucleic acid or rAAV is administered to cell line neurons. In some embodiments, the neurons are GABAergic neurons.

In some embodiments, administration of the isolated nucleic acid or rAAV results in at least a 2-fold, at least 10-fold, at least 20-fold, at least 20-fold increase in target gene expression (eg, SCN1A expression) relative to individuals not administered the isolated nucleic acid or rAAV 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times. In some embodiments, administration of the isolated nucleic acid or rAAV results in at least a 2-fold increase in target gene expression (eg, SCN1A expression) relative to target gene expression (eg, SCN1A) in the individual prior to administration of the isolated nucleic acid or rAAV times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.

In some aspects, the invention provides a method of modulating gene expression (eg, expression of SCN1A) in an individual, wherein an isolated nucleic acid or rAAV as described herein is administered to the individual expressing a target gene. In some embodiments, the expression of the target gene is abnormal (eg, increased or decreased) in an individual relative to a healthy individual. In some embodiments, the individual is haploinsufficient or suspected of haploinsufficiency relative to a healthy individual for the expression of the target gene.

In some embodiments, the individual has or is suspected of having a disease or disorder caused by a haploinsufficiency expression of the target gene. For example, in some embodiments, individuals with SCN1A haploinsufficiency suffer from Dravet syndrome. In some embodiments, the isolated nucleic acid or rAAV is administered to the individual by intravenous injection, intramuscular injection, inhalation, subcutaneous injection, and/or intracranial injection.

In some aspects, the present invention provides compositions comprising an isolated nucleic acid or rAAV as described by the present invention. In some embodiments, the composition includes a pharmaceutically acceptable carrier.

In some aspects, the present invention provides kits comprising a container containing an isolated nucleic acid or rAAV as described by the present invention. In some embodiments, the kit includes a container containing a pharmaceutically acceptable carrier. In some embodiments, the isolated nucleic acid or rAAV and the pharmaceutically acceptable carrier are contained in the same container. In some embodiments, the container is a syringe.

In some aspects, the present invention provides host cells comprising an isolated nucleic acid or rAAV as described by the present invention. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell line is a human cell, optionally a neuron, eg, a GABAergic neuron.

related applications

This application claims the benefit of the filing date of U.S. Provisional Application No. 63/056,528, filed July 24, 2020, entitled "DNA BINDING DOMAIN TRANSACTIVATORS AND USES THEREOF," which is incorporated by reference in its entirety. into this article.

Aspects of the invention relate to methods and compositions for modulating (eg, increasing) the expression of a target gene in a cell or individual, wherein the target gene is haploinsufficient (ie, the target gene comprises one functional copy). In some embodiments, the target gene is SCN1A.

In some embodiments, the present invention provides fusion proteins comprising a DNA binding domain, such as a ZFP, and a transcriptional regulatory domain. In some embodiments, the present invention provides fusion proteins comprising a DNA binding domain (such as a ZFP) and a transactivator domain (eg, a VPR domain). In some embodiments, the DNA binding protein binds to a target gene or a sequence of a regulatory region of a target gene. In some embodiments, the regulatory region is an enhancer sequence, a promoter sequence, or a suppressor sequence. In some embodiments, the promoter sequence can be an internal promoter (eg, located in an intron of the target gene) or an external promoter (eg, located upstream of the transcription start site of the target gene). In some embodiments, the DNA binding domains of the fusion proteins described herein bind to conserved sequences in the promoter regions of target genes (eg, SCN1A), and thus the transactivator domains increase gene expression.

In some aspects, the invention pertains to methods for increasing the expression of a target gene (eg, SCN1A) in a cell or individual. In some embodiments, the target gene contains a mutation that renders a cell or individual haploinsufficient for the target gene. Thus, in some embodiments, the methods and compositions of the present invention can be used to treat diseases and disorders associated with haploinsufficiency of target gene products (eg, Zoffer syndrome), which are typically caused by potential-gated sodium channels alpha times Caused by a mutation in one copy of the SCN1A gene of the unit Nav1.1 haploinsufficiency.

Transactivator fusion protein Some aspects of the invention relate to fusion proteins comprising a DNA binding domain (DBD) and a transactivator domain. As used herein, a fusion protein comprises two or more linked polypeptides encoded by two or more different amino acid sequences. As used herein, a chimeric protein is a fusion protein in which the two or more linked genes are from different species. Fusion proteins are typically produced recombinantly, wherein the gene encoding the fusion protein is in a system that supports the expression of the two or more linked genes and translates the resulting mRNA into a recombinant protein. In some embodiments, fusion proteins are recombinantly produced in prokaryotic or eukaryotic cells. Fusion proteins can be arranged in a variety of configurations. For example, one protein (Protein A) is located upstream of a second protein (Protein B). In other fusion protein configurations, protein B is located upstream of protein A. In some embodiments, the nucleic acid sequence encoding the DNA binding domain is located upstream of the nucleic acid sequence encoding the transactivator domain, and a fusion protein comprising the DBD linked to the transactivator is produced. In some embodiments, the nucleic acid sequence encoding the transactivator domain is located upstream of the nucleic acid sequence encoding the DNA binding domain, and a fusion protein comprising the transactivator domain linked to the DNA binding domain is produced. In some embodiments, the fusion protein comprises a transactivator domain upstream of the DNA binding domain. In some embodiments, the fusion protein comprises a DNA binding domain upstream of the transactivator domain.

In some embodiments, the fusion proteins described by the present invention comprise a DNA binding domain. As used herein, a "DNA binding domain (DBD)" refers to an independently folded protein comprising at least one structural motif that recognizes double- or single-stranded DNA (dsDNA or ssDNA). Certain DBDs recognize specific sequences (recognition sequences or motifs), while other types of DBDs have a general affinity for DNA. In some embodiments, the fusion proteins described by the present invention comprise sequence-specific DBDs. In some embodiments, the DBD recognizes (eg, specifically binds to) a nucleic acid sequence within or adjacent to a gene encoding a SCN1A protein (eg, Nav1.1). DBD-containing proteins are often involved in cellular processes such as transcription, replication, repair, and DNA storage. DBDs in transcription factors recognize specific DNA sequences in promoter regions or enhancer elements to facilitate gene expression. The transcription factor DBD is used as a fusion protein in genetic engineering to modulate target gene expression and can be mutated to alter DNA binding specificity or DNA binding affinity and thus modulate the expression of a desired target gene. Examples of DBDs include, but are not limited to, helix-turn-helix motifs, zinc finger motifs (including Cys2-His2 zinc fingers), transcription activator-like effectors (TALEs), winged helix motifs, HMG cassettes, dCas proteins ( For example, dCas9 or dCas12a), the homeodomain and the OB fold domain.

In some embodiments, the invention relates to zinc finger DBD fusion proteins. As used herein, a "zinc finger protein (ZFP)" refers to a protein containing at least one structural motif characterized by the coordination of one or more zinc ions that stabilize the protein fold. Zinc fingers are one of the most diverse structural motifs found in proteins, and as many as 3% of human genes encode zinc fingers. Most ZFPs contain multiple zinc fingers that make tandem contacts with target molecules, including DNA, RNA, and small proteins ubiquitin. "Classic" zinc finger motifs are composed of 2 cysteine amino acids and 2 histidine amino acids (C ₂ H ₂ ) and bind DNA in a sequence-specific manner. These ZFPs, including transcription factor IIIA (TFIIIA), are often involved in gene expression. Multiple zinc finger motifs in DNA-binding proteins bind and wrap around the outside of the DNA double helix. Zinc finger domain fusion proteins can be used to generate DBDs with novel DNA binding specificities due to their relatively small size (eg, about 25 to 40, typically 27 to 35 amino acids per finger). These DBDs can deliver other fusion domains (eg, transcriptional activation or repression domains or epigenetic modification domains) to alter transcriptional regulation of target genes. In some embodiments, the zinc finger protein comprises 2 to 8 fingers, wherein each finger contains 27 to 40 amino acids (eg, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids).

In some embodiments, the ZFP comprises 1, 2, 3, 4, 5, 6, 7 or 8 zinc fingers. Each zinc finger may contain 25 to 40, 25 to 30, 30 to 35, 35 to 40, or 40 to 45 amino acids. In some embodiments, the zinc fingers comprise 27 to 35 amino acids. In some embodiments, the zinc finger comprises 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids. Zinc fingers can specifically recognize or bind to a haploinsufficient target sequence in an individual, eg, a target gene or a regulatory region of a target gene. In some embodiments, the zinc finger binds to, eg, a target sequence of the SCN1A gene (eg, human SCN1A) as set forth in SEQ ID NO: 49. In some embodiments, the zinc finger that binds to the target sequence of the SCN1A gene comprises one or more amino acid sequences of SEQ ID NOs: 63 to 80, or a combination thereof. In some embodiments, the zinc finger specifically recognizes or binds to a target sequence comprising a trinucleotide sequence.

In some embodiments, the zinc finger comprises a recognition helix that recognizes or binds to a target sequence (eg, a target sequence comprising a trinucleotide sequence). In some embodiments, the recognition helix is bound to a trinucleotide. In some embodiments, the recognition helix comprises 4 to 10 amino acids. In some embodiments, the recognition helix comprises 4, 6, 7, 8, 9 or 10 amino acids. In some embodiments, the recognition helix binds to the trinucleotide sequence of the SCN1A gene. In some embodiments, the recognition sequence bound to the SCN1A gene comprises the amino acid sequence of any one of SEQ ID NOs: 17-22, 29-34, or 41-46. In some embodiments, the recognition sequence that binds to the SCN1A gene is encoded by any of SEQ ID NOs: 11-16, 23-28, or 35-40. In some embodiments, the zinc finger binds to the same nucleotide sequence as the recognition helix comprising the amino acid sequence of any of SEQ ID NOs: 17-22, 29-34, or 41-46.

In some embodiments, a zinc finger comprises a linker sequence at its C-terminus that can be used to link or link the zinc finger to another zinc finger. In some embodiments, the linker sequence can be a typical linker, eg, an amino acid sequence comprising TGEKP (SEQ ID NO: 120). In some embodiments, the linker sequence can be an atypical linker, eg, an amino acid sequence comprising TGSQKP (SEQ ID NO: 121). In some embodiments, the linker sequence can be 2 to 10 amino acids, eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

In some embodiments, a ZFP that binds to a target gene (eg, SCN1A gene) comprises six zinc fingers, each of which recognizes or binds to a different trinucleotide sequence of the target gene (eg, SCN1A gene) . In some embodiments, the ZFP that binds to the SCN1A gene comprises the amino acid sequence of SEQ ID NO:57. In some embodiments, the ZFP that binds to the SCN1A gene comprises a zinc finger comprising the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67 and/or 68. In some embodiments, the ZFP that binds to the SCN1A gene includes a recognition helix comprising the amino acid sequence of SEQ ID NO: 17, 18, 19, 20, 21 and/or 22. In some embodiments, the ZFP that binds to the SCN1A gene comprises the amino acid sequence of SEQ ID NO:59. In some embodiments, the ZFP that binds to the SCN1A gene includes a zinc finger comprising the amino acid sequence of SEQ ID NO: 69, 70, 71, 72, 73 and/or 74. In some embodiments, the ZFP that binds to the SCN1A gene includes a recognition helix comprising the amino acid sequence of SEQ ID NOs: 29, 30, 31, 32, 33 and/or 34. In some embodiments, the ZFP that binds to the SCN1A gene comprises the amino acid sequence of SEQ ID NO:61. In some embodiments, the ZFP that binds to the SCN1A gene comprises a zinc finger comprising the amino acid sequence of SEQ ID NO: 75, 76, 77, 78, 79 and/or 80. In some embodiments, the ZFP that binds to the SCN1A gene includes a recognition helix comprising the amino acid sequence of SEQ ID NOs: 41, 42, 43, 44, 45, and/or 46. In some embodiments, the ZFP that binds to the SCN1A gene comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% of SEQ ID NO: 57, 59 or 61 as shown below %, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity.

SEQ ID NO: 57 ( amino acid sequence of ZFP1 protein ) RPFQCRICMRNFSQRGNLVRHIRTHTGEKPFACDICGKKFALSFNLTRHTKIHTGSQKPFQCRICMRNFSRSDNLTRHIRTHTGEKPFACDICGKKFADRSHLARHTKIHTGSQKPFQCRICMRNFSQKAHLTAHIRTHTGEKPFACDICGRKFARSDNLTRHTKIHLRQKD

SEQ ID NO: 59 ( amino acid sequence of ZFP2 protein ) RPFQCRICMRNFSRSSNLTRHIRTHTGEKPFACDICGKKFADKRTLIRHTKIHTGSQKPFQCRICMRNFSQRGNLVRHIRTHTGEKPFACDICGKKFALSFNLTRHTKIHTGSQKPFQCRICMRNFSRSDNLTRHIRTHTGEKPFACDICGRKFADRSHLARHTKIHLRQKD

SEQ ID NO: 61 ( amino acid sequence of ZFP3 protein ) RPFQCRICMRNFSDRSALARHIRTHTGEKPFACDICGKKFARSDNLTRHTKIHTGSQKPFQCRICMRNFSQSGDLTRHIRTHTGEKPFACDICGKKFAVRQTLKQHTKIHTGSQKPFQCRICMRNFSAAGNLTRHIRTHTGEKPFACDICGRKFARSDNLTRHTKIHLRQKD

In some embodiments, the DBD is a transcriptional activator-like effector protein (TALE). A TALE can specifically recognize or bind to a target sequence, eg, a target gene or a regulatory region of a target gene. In some embodiments, each system is haploinsufficient for the target gene. In some embodiments, the TALE binds to the target sequence of the SCN1A gene (eg, human SCN1A as provided in SEQ ID NO: 49). TALE proteins are secreted by bacteria and bind to promoter sequences in host plants to activate the expression of plant genes that facilitate bacterial infection. Typically, TALE proteins are manipulated to bind novel DNA sequences, since target sequences are recognized by a central repeat domain consisting of a variable number of ~30 to 35 amino acid repeats, each of which recognizes a single base within the target sequence right. Arrays of these repeats are often necessary to identify DNA sequences.

In some embodiments, the DBD is a homology domain. A homology domain can specifically recognize or bind to a target sequence, eg, a target gene or a regulatory region of a target gene. In some embodiments, each system is haploinsufficient for the target gene. In some embodiments, the homeodomain binds to the target sequence of the SCN1A gene (eg, human SCN1A as provided in SEQ ID NO: 49). Homeodomains are proteins containing three alpha helices and an N-terminal arm responsible for recognizing target sequences. Homeodomains typically recognize small DNA sequences (~4 to 8 base pairs), however these domains can be tandemly fused to other DNA-binding domains (other homeodomains or zinc finger proteins) to recognize longer stretches (12 to 24 base pairs). Thus, the homeodomain may be a DBD component that recognizes a unique sequence within the human genome.

In some embodiments, at least one DNA binding domain is a catalytically inactive CRISPR-associated protein (Cas protein). Catalytically inactive Cas proteins (also known as dCas or "dead Cas proteins") are those that have been modified or mutated such that nuclease activity (eg, endonuclease activity) is reduced or lacks all nuclease activity (eg, endonuclease activity) activity) of the Cas protein. In some embodiments, the catalytically inactive Cas protein is a dCas9 or dCas12 protein. In some embodiments, the DBD is a dCas protein (also known as "dead Cas"), such as dCas9 or dCas12a. dCas proteins are mutant variants of CRISPR-associated proteins (Cas, eg, Cas9 or Cas12a) that have been mutated to render them inactive (ie, unable to cleave nucleotides). dCas can specifically recognize or bind to a target sequence, eg, a target gene or a regulatory region of a target gene. A complex comprising a dCas protein and a guide nucleic acid (eg, gRNA) can target and/or bind to a specific nucleotide sequence or gene complementary to the guide nucleic acid. In some embodiments, each system target gene is haploinsufficient. In some embodiments, the dCas binds to the target sequence of the SCN1A gene (eg, human SCN1A as provided in SEQ ID NO: 49). However, when bound to a guide nucleic acid (eg, guide RNA, gRNA, or sgRNA) that is complementary or partially complementary to the target DNA sequence, dCas proteins retain their ability to recognize and bind to the target DNA sequence. In some embodiments, the guide nucleic acid used to target a dCas (eg, dCas9) protein to SCN1A comprises a spacer sequence having any of SEQ ID NOs: 85, 86, 89, 90, 93 or 94. In some embodiments, the guide nucleic acid for targeting a dCas (eg, dCas9) protein to SCN1A comprises at least 15 (eg, at least 15) of any of SEQ ID NOs: 85, 86, 89, 90, 93, or 94 16, 17, 18, 19 or 20) spacer sequences of consecutive nucleotides. In some embodiments, the guide nucleic acid for targeting a dCas (eg, dCas9) protein to SCN1A comprises any one of SEQ ID NOs: 83, 84, 87, 88, 91 or 92. In some embodiments, the guide nucleic acid for targeting a dCas (eg, dCas9) protein to SCN1A comprises or consists of any one of SEQ ID NOs: 83-94. Thus, the dCas endonuclease may be a DBD component that recognizes a unique sequence within the human genome. In some embodiments, the fusion protein comprises a dCas9 protein and a transactivation domain (eg, a VPR domain).

In some aspects, the invention relates to DNA binding domains that bind to genes encoding potential-gated sodium channels (eg, _Nav 1.1). In some embodiments, the gene encoding the potential-gated sodium channel is the SCN1A gene and comprises the sequence set forth in SEQ ID NO:49. In some embodiments, the DNA binding domain binds to an untranslated region of a target gene, such as a 3'-untranslated region (3'UTR) or a 5'-untranslated region (5'UTR). In some embodiments, the non-translated region comprises regulatory sequences, such as enhancer, promoter, intron or suppressor sequences. In some embodiments, the DNA binding domain is a zinc finger protein comprising the sequences set forth in SEQ ID NOs: 57-62. In some embodiments, the DNA binding domain binds to the nucleic acid sequence set forth in any one of SEQ ID NOs: 5-7. In some embodiments, the DNA binding domain binds to the full length of the nucleic acid sequence set forth in any one of SEQ ID NOs: 5-7. In some embodiments, the DNA binding domain binds to at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 of the nucleic acid sequences set forth in any one of SEQ ID NOs: 5-7 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 consecutive nucleotides. In some embodiments, the DNA binding domain binds to the nucleic acid sequence set forth in SEQ ID NO:3. In some embodiments, the DNA binding domain binds to at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 of the nucleic acid sequence set forth in any one of SEQ ID NO: 3 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 consecutive nucleotides.

SEQ ID NO: 3 , SCN1A targeting region, nucleotide sequence TTTTTTTTTTTTTTTTTGAAACAAGCTATTTGCTGATTTGTATTAGGTACCATAGAGTGAGGCGAGGATGAAGCCGAGAGGATACTGCAGAGGTCTCTGGTGCATGTGTGTATGTGTGCGTTTGTGTGTG

The number of DNA binding domains encoded by the transgene can vary. In some embodiments, the transgene encodes a DNA binding domain. In some embodiments, the transgene encodes two DNA binding domains. In some embodiments, the transgene encodes three DNA binding domains. In some embodiments, the transgene encodes four DNA binding domains. In some embodiments, the transgene encodes five DNA binding domains. In some embodiments, the transgene encodes six DNA binding domains. In some embodiments, the transgene encodes seven DNA binding domains. In some embodiments, the transgene encodes 8 DNA binding domains. In some embodiments, the transgene encodes nine DNA binding domains. In some embodiments, the transgene encodes 10 DNA binding domains. In some embodiments, the transgene encodes more than 10 (eg, 20, 30, 50, 100, etc.) DNA binding domains. The DNA binding domains can be the same DNA binding domain (eg, multiple copies of the same DBD), different DNA binding domains (eg, each DBD binds a unique sequence), or a combination thereof.

In some aspects, the invention relates to fusion proteins comprising transactivator domains. As used herein, a "transactivation domain" refers to a scaffold domain in a transcription factor that contains binding sites for other proteins that regulate gene expression, such as transcriptional coregulators. In some embodiments, a transactivation domain (also referred to as a transcription activation domain) is used in conjunction with DBD to activate transcription from a promoter or enhancer, either directly via contact with a transcription factor, or indirectly via a coactivator protein. Transactivation domains (TADs) are often named for their amino acid composition, which are either essential for activity or the most abundant in TADs. TAD is used as a fusion protein in genetic engineering to modulate the expression of a target gene and can be mutated to alter the degree of transcriptional activation and thus the expression of the target gene. Examples of transactivation domains include, but are not limited to, GAL4, HAP1, VP16, P65, RTA, GCN4, TCF4 AD1, TCF4 AD2, MEF2A, MEF2C, MEF2D, Sp1 glutamate-rich domain, p53, E2F1, MyoD, MAPK7, NF1B proline-rich domain, RelA and HSF1.

In some embodiments, the transactivation subdomain comprises a VP64 domain. VP64 is an acidic TAD consisting of four tandem copies of the VP16 protein, which is naturally expressed by the herpes simplex virus. When fused to DBD bound at or near the promoter of a gene, VP64 acts as a strong transcriptional activator and can thus be used to regulate the expression of target genes (eg, SCN1A). The VP64 domain typically consists of a tetrameric repeat of the minimal activation domain of the herpes simplex protein VP16. In some embodiments, the VP64 domain comprises four repeats of amino acid residues 437 to 448 in VP16. In some embodiments, the VP16 protein is encoded by the human herpesvirus 2 UL48 gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NC_001798.2. In some embodiments, the VP16 gene comprises 99% identity, 95% identity, 90% identity, 80% identity to the amino acid sequence encoded by the nucleic acid sequence set forth in NCBI Reference Sequence Accession No.: YP_009137200.1 Nucleotide sequences that are identical, 70% identical, 60% identical or 50% identical. In some embodiments, the VP16 protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in NCBI Reference Sequence Accession No. Q69113-1 , 60% identical or 50% identical amino acid sequences. In some embodiments, the VP16 gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 51 Nucleotide sequences that are identical, 60% identical, or 50% identical. In some embodiments, the VP16 protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the amino acid sequence set forth in SEQ ID NO: 52 Amino acid sequences that are identical or 50% identical.

In some embodiments, the transactivator subdomain comprises a p65 activation domain. P65 is a subunit of NF-κβ transcription factor, and its C-terminus contains two adjacent acidic TADs. When fused to a DBD bound at or near the promoter of a gene, the p65 protein acts as a strong transcriptional activator and can thus be used to regulate the expression of target genes, for example as described by Urlinger et al., "The p65 domain from NF-kappaB is an efficient human activator in the tetracycline-regulatable gene expression system”, described in Gene, 2000. In some embodiments, the p65 protein is encoded by the human RELA gene comprising the sequence set forth in NCBI Reference Sequence Accession Nos.: NM_001145138.1, NM_001243984.1, NM_001243985.1, or NM_021975.3. In some embodiments, the RELA gene comprises 99% of the amino acid sequence encoded by the nucleic acid sequence set forth in any of NCBI Reference Sequence ID NOs: NM_001145138.1, NM_001243984.1, NM_001243985.1, or NM_021975.3 Nucleotide sequences that are identical, 95% identical, 90% identical, 80% identical, 70% identical, 60% identical or 50% identical. In some embodiments, the p65 protein comprises 99% identity, 95% identity, 90% identity, 80% identity to the amino acid sequences set forth in NP_001138610.1, NP_001230913.1, NP_001230914.1, and NP_068110.3 Amino acid sequences that are identical, 70% identical, 60% identical, or 50% identical. In some embodiments, the RELA gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 53 Nucleotide sequences that are identical, 60% identical, or 50% identical. In some embodiments, the p65 protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the amino acid sequence set forth in SEQ ID NO: 54 Amino acid sequences that are identical or 50% identical.

In some embodiments, the transactivator subdomain comprises an RTA domain. RTA is a hydrophobic TAD derived from Epstein Barr virus, a potent transactivation domain that binds to enhancer regions to facilitate the expression of several viral genes. When fused to DBD bound at or near the promoter of a gene, the RTA protein acts as a strong transcriptional activator and can thus be used to regulate the expression of target genes, as described, for example, by Miyazawa et al., "IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3", described in Journal of Biological Chemistry, 2018. In some embodiments, the RTA protein is encoded by the Epsteinbarr virus BRLF1 gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: YP_041674.1. In some embodiments, the BRLF1 gene comprises 99% identity, 95% identity, 90% identity to the amino acid sequence encoded by any one of the nucleic acid sequences set forth in NCBI Reference Sequence ID NO: YP_041674.1 , 80% identical, 70% identical, 60% identical or 50% identical nucleotide sequences. In some embodiments, the RTA protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the amino acid sequence set forth in YP_041674.1 or 50% identical amino acid sequences. In some embodiments, the BRLF1 gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 55 Nucleotide sequences that are identical, 60% identical, or 50% identical. In some embodiments, the RTA protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the amino acid sequence set forth in SEQ ID NO: 56 Amino acid sequences that are identical or 50% identical.

In some embodiments, the transactivator domain comprises a transcription factor 4 (TCF4) activation domain. In some embodiments, the TCF4 activation domain is a protein domain of the TCF4 protein. In some embodiments, the TCF4 protein is encoded by the human TCF4 gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_003199. In some embodiments, the TCF4 gene comprises 99% identity, 95% identity, 90% identity, 80% identity to the amino acid sequence encoded by the nucleic acid sequence set forth in any one of NCBI Reference Sequence ID NO: NM_003199 % identical, 70% identical, 60% identical or 50% identical nucleotide sequences. In some embodiments, the TCF4 protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the amino acid sequence set forth in NP_003190.1 or 50% identical amino acid sequences. In some embodiments, the TCF4 activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 122 % identical, 60% identical or 50% identical amino acid sequences. In some embodiments, the TCF4 activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 123 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 122 , TCF4 activation domain 1 , amino acid sequence MHHQQRMAALGTDKELSDLLDFSAMFSPPVSSGKNGPTLASGHFTGSNVEDRSSSGSWGNGGHPSPSRNYGDGTPYDHMTSRDLGSHDNLSPPFVNS

SEQ ID NO: 123 , TCF4 activation domain 2 , amino acid sequence TNNSFSSNPSTPVGSPPSLSAGTAVWSRNGGQASSSPNYEGPLHSLQSRIEDRLERLDDAIHVLRNHAVGPS

In some embodiments, the transactivator domain comprises a myocyte-specific enhancer factor 2A (MEF2A) activation domain. In some embodiments, the MEF2A activation domain is a protein domain of the MEF2A protein. In some embodiments, the MEF2A protein is encoded by the human MEF2A gene comprising the sequence set forth in any of NCBI Reference Sequence Accession Nos.: NM_001130926.2, NM_001130927.3, or NM_001130928.2. In some embodiments, the MEF2A protein comprises an amino acid sequence that is 99% identical to an amino acid sequence encoded by any of the nucleic acid sequences set forth in NCBI Reference Sequence ID NO: NM_001130926.2, NM_001130927.3, or NM_001130928.2, 95 Sequences of % identity, 90% identity, 80% identity, 70% identity, 60% identity or 50% identity. In some embodiments, the MEF2A protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70 % identical, 60% identical or 50% identical amino acid sequences. In some embodiments, the MEF2A activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 124 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 124 , MEF2A activation domain, amino acid sequence PLSEEEELELNTQR

In some embodiments, the transactivator domain comprises a myocyte enhancer factor 2C (MEF2C) activation domain. In some embodiments, the MEF2C activation domain is a protein domain of the MEF2C protein. In some embodiments, the MEF2C protein is encoded by the human MEF2C gene comprising the sequence set forth in any of NCBI Reference Sequence Accession Nos.: NM_001131005.2, NM_001193347.1, NM_001193348.1, or NM_001193349.2. In some embodiments, the MEF2C gene comprises 99% identity, 95% identity to the nucleic acid sequence set forth in any of NCBI Reference Sequence ID NOs: NM_001131005.2, NM_001193347.1, NM_001193348.1 or NM_001193349.2 , 90% identity, 80% identity, 70% identity, 60% identity or 50% identity nucleotide sequences. In some embodiments, the MEF2C protein comprises 99% identity, 95% identity to the amino acid sequence set forth in any one of NCBI Reference Sequence ID NOs: NP_001124477.1, NP_001180276.1, NP_001180277.1, or NP_001180278.1 Amino acid sequences that are identical, 90% identical, 80% identical, 70% identical, 60% identical, or 50% identical. In some embodiments, the MEF2C activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 125 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 125 , MEF2C activation domain, amino acid sequence SVSEDVDLLLNQR

In some embodiments, the transactivator domain comprises a myocyte enhancer factor 2D (MEF2D) activation domain. In some embodiments, the MEF2D activation domain is a protein domain of a MEF2D protein. In some embodiments, the MEF2D protein is encoded by the human MEF2D gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_001271629.2 or NM_005920.4. In some embodiments, the MEF2D gene comprises 99% identity, 95% identity, Nucleotide sequences that are 90% identical, 80% identical, 70% identical, 60% identical or 50% identical. In some embodiments, the MEF2D protein comprises 99% identity, 95% identity, 90% identity, Amino acid sequences of 80% identity, 70% identity, 60% identity or 50% identity. In some embodiments, the MEF2D activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 126 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 126 , MEF2D activation domain, amino acid sequence HLTEDHLDLNNAQR

In some embodiments, the transactivator domain comprises the transcription factor Sp1 glutamate-rich activation domain. In some embodiments, the activation domain is a protein domain of a protein. In some embodiments, the protein is encoded by the human SP1 gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_001251825.2. In some embodiments, the gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the nucleic acid sequence set forth in NCBI Reference Sequence ID No: NM_001251825.2 % identical or 50% identical nucleotide sequences. In some embodiments, the protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the amino acid sequence set forth in NCBI Reference Number: NP_001238754.1 % identical or 50% identical amino acid sequences. In some embodiments, the transcription factor Sp1 glutamate-rich activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, Amino acid sequences of 80% identity, 70% identity, 60% identity or 50% identity.

SEQ ID NO: 127 ， SP1 富麩胺酸活化域，胺基酸序列NSVSAATLTPSSQAVTISSSGSQESGSQPVTSGTTISSASLVSSQASSSSFFTNANSYSTTTTTSNMGIMNFTTSGSSGTNSQGQTPQRVSGLQGSDALNIQQNQTSGGSLQAGQQKEGEQNQQTQQQQILIQPQLVQGGQALQALQAAPLSGQTFTTQAISQETLQNLQLQAVPNSGPIIIRTPTVGPNGQVSWQTLQLQNLQVQNPQAQTITLAPMQGVSLGQTSSSNTTLTPIA

In some embodiments, the transactivator domain comprises a tumor protein p53 activation domain. In some embodiments, the p53 activation domain is a protein domain of the p53 protein. In some embodiments, the p53 protein is encoded by the human p53 gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_000546.6 or NM_001126112.2. In some embodiments, the p53 gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the nucleic acid sequence set forth in NCBI Reference Sequence ID No: NM_000546.6 or NM_001126112.2 % identical, 60% identical or 50% identical nucleotide sequences. In some embodiments, the p53 protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70 % identical, 60% identical or 50% identical amino acid sequences. In some embodiments, the p53 activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 128 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 128 , p53 activation domain, amino acid sequence MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLS

In some embodiments, the transactivator domain comprises an E2F transcription factor 1 (E2F1) activation domain. In some embodiments, the E2F transcription factor 1 activation domain is the protein domain of the E2F transcription factor 1 protein. In some embodiments, the E2F transcription factor 1 protein is encoded by the human E2F1 gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_005225.3. In some embodiments, the E2F1 gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, Nucleotide sequences that are 60% identical or 50% identical. In some embodiments, the E2F1 protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, Amino acid sequences with 60% identity or 50% identity. In some embodiments, the E2F1 activation domain comprises amino acid residues 380 to 437 of the E2F1 protein having the amino acid sequence set forth in NCBI reference number: NP_005216.1. In some embodiments, the E2F1 activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 129 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 129 , E2F transcription factor 1 activation domain, amino acid sequence ADSLLEHVREDFSGLLPEEFISLSPPHEALDYHFGLEEGEGIRDLFDCDFGDLTPLDF

In some embodiments, the transactivator domain comprises a myoblast assay protein 1 (MyoD) activation domain. In some embodiments, the MyoD activation domain is the protein domain of the MyoD protein. In some embodiments, the MyoD protein is encoded by the human MyoD gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_002478.5. In some embodiments, the MyoD gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, nucleotide sequence set forth in NCBI Reference Sequence ID No: NM_002478.5, Nucleotide sequences that are 60% identical or 50% identical. In some embodiments, the MyoD protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, Amino acid sequences with 60% identity or 50% identity. In some embodiments, the MyoD activation domain comprises amino acid residues 1 to 63 of the MyoD protein having the amino acid sequence set forth in NCBI reference number: NP_002469.2. In some embodiments, the MyoD activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 130 % identical, 60% identical or 50% identical amino acid sequences.

yo SEQ ID NO: 130 , MyoD activation domain, amino acid sequence MELLSPPLRDVDLTAPDGSLCSFATTDDFYDDPCFDSPDLRFFEDLDPRLMHVGALLKPEEHS

In some embodiments, the transactivator domain comprises a mitogen-activated protein kinase 7 (MAPK7) activation domain. In some embodiments, the MAPK7 activation domain is the protein domain of the MAPK7 protein. In some embodiments, the MAPK7 protein is encoded by the human MAPK7 gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_002749.4. In some embodiments, the MAPK7 gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, a nucleic acid sequence set forth in NCBI Reference Sequence ID No: NM_002749.4, Nucleotide sequences that are 60% identical or 50% identical. In some embodiments, the MAPK7 protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, Amino acid sequences with 60% identity or 50% identity. In some embodiments, the MAPK7 activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 131 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 131 , MAPK7 activation domain, amino acid sequence LAAQSLVPPPGLPGSSTPGVLPYFPPGLPPPDAGGAPQSSMSESPDVNLVTQQLSKSQVEDPLPPVFSGTPKGSGAGYGVGFDLEEFLNQSFDMGVADGPQDGQADSASLSASLLADWLEGHGMNPA

In some embodiments, the transactivator domain comprises a nuclear factor 1 type B (NF1B) proline-rich activation domain. In some embodiments, the NF1B proline-rich activation domain is a protein domain of the NF1B protein. In some embodiments, the NF1B protein is encoded by the human NF1B gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_001369480. In some embodiments, the NF1B gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the nucleic acid sequence set forth in NCBI Reference Sequence ID No: NM_001369480 Nucleotide sequences that are identical or 50% identical. In some embodiments, the NF1B protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, Amino acid sequences with 60% identity or 50% identity. In some embodiments, the NF1B activation domain comprises amino acid residues 319 to 419 of the NF1B protein having the amino acid sequence set forth in NCBI reference number: NP_001356409.1. In some embodiments, the NF1B activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 132 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 132 , NF1B proline-rich activation domain, amino acid sequence PEKPLFSSASPQDSSPRLSTFPQHHHPGIPGVAHSVISTRTPPPPSPLPFPTQAILPPAPSSYFSHPTIRYPPHLNPQDTLKNYVPSYDPSSPQTSQSWYLG

In some embodiments, the transactivator subdomain comprises a RelA activation domain. In some embodiments, the RelA activation domain is the protein domain of the RelA protein. In some embodiments, the RelA protein is encoded by the human RelA gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_001145138.2 or NM_021975.4. In some embodiments, the RelA gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the nucleic acid sequence set forth in NCBI Reference Sequence ID No: NM_001145138.2 or NM_021975.4 % identical, 60% identical or 50% identical nucleotide sequences. In some embodiments, the RelA protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70 % identical, 60% identical or 50% identical amino acid sequences. In some embodiments, the RelA activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 133 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 133 ， R elA 活化域，胺基酸序列QYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALL

In some embodiments, the transactivator domain comprises a heat shock transcription factor 1 (HSF1) activation domain. In some embodiments, the HSF1 activation domain is a protein domain of the HSF1 protein. In some embodiments, the HSF1 protein is encoded by the human HSF1 gene comprising the sequence set forth in NCBI Reference Sequence Accession No.: NM_005526.4. In some embodiments, the HSF1 gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, Nucleotide sequences that are 60% identical or 50% identical. In some embodiments, the HSF1 protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, Amino acid sequences with 60% identity or 50% identity. In some embodiments, the HSF1 activation domain comprises 100% identity, 99% identity, 95% identity, 90% identity, 80% identity, 70% identity to the amino acid sequence set forth in SEQ ID NO: 134 % identical, 60% identical or 50% identical amino acid sequences.

SEQ ID NO: 134 , HSF1 activation domain, amino acid sequence GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS

The present invention is based in part on fusion proteins comprising hybrid transactivator domains. As used herein, a "hybrid transactivator domain" refers to a fusion protein comprising more than one transcriptional activator protein or portion thereof (eg, 2, 3, 4, 5 or more transcriptional activator proteins, or portions thereof). Hybrid transactivation domains are used in genetic engineering to increase the expression of target genes. In some embodiments of the invention, as described in Chavez et al., using a VP64-P65-RTA (VPR) containing VP64-P65-RTA (VPR) as described in "Highly efficient Cas9-mediated transcriptional programming", Nat Methods, 2015, (SEQ ID NO: 47) Triple hybrid transactivation domains of nucleotide sequences to increase target gene (eg SCN1A) expression.

In some embodiments, the fusion proteins described herein can comprise a DBD (eg, ZFP) and a transcriptional repressor protein. In some aspects, the invention relates to fusion proteins comprising transcriptional repressor domains. As used herein, a "transcriptional repressor" protein generally refers to a polypeptide that downregulates the expression of a target gene. Examples of transcriptional repressors include, but are not limited to, KRAB, SMRT/TRAC-2, and NCoR/RIP-13. In some embodiments, these transcriptional repressor fusion proteins are useful for reducing the expression of target genes (eg, genes overexpressed in gain-of-function diseases).

In some embodiments, the fusion proteins described herein additionally comprise a nuclear localization signal or sequence (NLS). NLS is an amino acid sequence that facilitates the entry of proteins into the nucleus. In some embodiments, the NLS comprises a plurality of amino acid sequences of positively charged amino acids (eg, lysine or arginine). In some embodiments, the NLS comprises any one of SEQ ID NOs: 135-140. In some embodiments, the NLS comprises one or more of SEQ ID NOs: 135-140 (eg, any combination). The NLS can be at the N-terminus or the C-terminus of the fusion proteins described herein. In some embodiments, the NLS can be located within the protein. Table A: Nuclear localization sequences identifier sequence SEQ ID NO: SV40 NLS PKKKRKVE 135 cMyc NLS PAAKRVKLD 136 cMyc-like NLS PAAKKKLD 137 nucleoplasmin NLS KRPAATKKAGQAKKKKLD 138 Duolian SV40 NLS KRTADGSEFESTPKKKRKVE 139 Duo TCF4 NLS PRRRPLHSSAMEVQTKKVRKVPP 140

isolated nucleic acid An isolated nucleic acid sequence refers to a DNA or RNA sequence. In some embodiments, the proteins and nucleic acids of the invention are isolated. As used herein, the term "isolated" means artificially generated. As used herein in reference to nucleic acids, the term "isolated" means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) produced by clonal recombination; (iii) as by Cleavage and gel separation purification; or (iv) synthesis by, for example, chemical synthesis. Isolated nucleic acids can be readily manipulated by recombinant DNA techniques well known in the art. Therefore, it is believed that the nucleotide sequence contained in the vector in which the 5' and 3' restriction sites are known or in which the polymerase chain reaction (PCR) primer sequence has been disclosed is isolated but exists in its natural state in its natural host The nucleic acid sequences in this have not been isolated. The isolated nucleic acid can be substantially purified, but need not be. For example, an isolated nucleic acid within a colonization or expression vector is not pure because the nucleic acid may contain only a small percentage of material in the cell in which it resides. However, this nucleic acid is isolated, as the term is used herein, because it can be readily manipulated by standard techniques known to those of ordinary skill. As used herein in reference to a protein or peptide, the term "isolated" refers to a protein or peptide that has been isolated from its natural environment or artificially produced (eg, by chemical synthesis, by recombinant DNA, etc.).

In some aspects, the invention relates to isolated nucleic acids (eg, expression constructs, such as rAAV vectors) configured to express one or more ZFP-transactivation domain fusion proteins. In some embodiments, the fusion protein comprises 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) DBDs and/or 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) transactivation subdomains. In some embodiments, the fusion protein comprises more than 10 DBS and/or more than 10 transactivator domains.

In some aspects of the invention, the DNA binding domain is fused to the transcriptional regulatory domain indirectly via a linker. As used herein, a "linker" is generally a stretch of polypeptide that structurally links two different polypeptides within a single transgene. In some embodiments, the linker is flexible to allow movement of different polypeptides. In some embodiments, the flexible linker comprises a glycine residue. In some embodiments, the flexible linker comprises a mixture of glycine and serine residues. In some embodiments, the linker is cleavable, allowing isolation of the polypeptide. In some embodiments, the cleavable linker is cleaved by a protease. In some embodiments, the protease is trypsin or factor X.

In some embodiments, the linker comprises 5 to 30 amino acids (eg, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids). In some embodiments, the linker comprises 3 to 30 amino acids (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids). In some embodiments, the linker comprises 3 to 20 amino acids (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids).

The present invention is based in part on fusion proteins engineered to increase the expression of genes (eg, SCN1A) encoding potential-gated sodium ion channel subunit proteins (also known as SCN proteins). As used herein, "SCN protein" refers to a sodium ion channel protein that mediates potential-dependent sodium ion permeability of excitatory membranes, allowing sodium ions to pass through the membrane. Examples of SCN proteins in humans include, but are not limited to, SCN1A, SCN3A, SCN5A, SCN10A, and SCN11A. In some embodiments, the SCN protein is SCN1A (also known as Nav1.1), which encodes the type 1 alpha ₁ ion channel subunit. In some embodiments, the SCN protein is the SCN1B protein, which encodes the type 1 beta ₁ ion channel subunit or the SCN1C protein. In some embodiments, the SCN protein is a combination of SCN1A, SCN1B and/or SCN1C proteins. As disclosed herein, an SCN protein can be a portion or fragment of an SCN protein. In some embodiments, the SCN proteins as disclosed herein are variants of the SCN proteins, such as point mutants or truncation mutants.

In humans, the SCN1A line is encoded by the SCN1A gene (Gene ID: 6323, human), which is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, rats and chickens. The SCN1A gene in humans is mainly expressed in the brain, lung and testis. In some embodiments, the SCN1A protein comprises five structural repeats (I, II, III, IV, Q).

In some embodiments, the SCN1A protein is encoded by the human SCN1A gene comprising NCBI Reference Sequence ID Nos: NM_001165963.2, NM_00165964.2, NM_001202435.2, NM_001353948.1, NM_001353949.1, NM_001353950.1, NM_001353950.1 1. The sequence set forth in NM_001353952.1, NM_001353954.1, NM_00353955.1, NM_001353957.1, NM_001353958.1, NM_001353960.1, NM_001353961.1 or NM_006920.5. In some embodiments, the SCN1A protein is encoded by the mouse SCN1A gene comprising the sequence set forth in NCBI Reference Sequence ID No: NM_001313997.1 or NM_018733.2. In some embodiments, the SCN1A protein comprises 99% identity, 95% identity, Amino acid sequences of 90% identity, 80% identity, 70% identity, 60% identity or 50% identity. In some embodiments, the SCN1A gene comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the sequence set forth in SEQ ID NO: 50, or 50% identical amino acid sequence. In some embodiments, the human SCN1A protein comprises NCBI Reference Sequence ID Nos: NP_001159435.1, NP_0011159436.1, NP_001189364.1, NP_001340877.1, NP_001340878.1, NP_001340870.1, NP3_001340880.1, NP_000138 1. Sequences set forth in NP_001340884.1, NP_001340886.1, NP_001340887.1, NP_001340889.1, NP_001340890.1, NP_00851.3. In some embodiments, the SCN1A protein comprises 99% identity, 95% identity, Amino acid sequences of 90% identity, 80% identity, 70% identity, 60% identity or 50% identity. In some embodiments, the mouse SCN1A protein comprises the sequence set forth in NCBI Reference Sequence ID No: NP_001300926.1 or NP_061203.2. In some embodiments, the human SCN1A protein comprises 99% identity, 95% identity, 90% identity, 80% identity, 70% identity, 60% identity to the nucleic acid sequence set forth in SEQ ID NO: 49 Amino acid sequences that are sexual or 50% identical.

The isolated nucleic acid of the present invention may be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, an isolated nucleic acid as described by the present invention includes a region (eg, a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof. The isolated nucleic acid (eg, a recombinant AAV vector) can be packaged within a capsid protein and administered to an individual and/or delivered to a target cell of choice. A "recombinant AAV (rAAV) vector" typically consists of at least a transgene and its regulatory sequences and 5' and 3' AAV inverted terminal repeats (ITRs). As described elsewhere herein, the transgene may comprise regions encoding, for example, proteins and/or expression control sequences (eg, polyadenylation tails).

Typically, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding these ITRs are used in the molecule, although some minor modifications to these sequences are permitted. The ability to modify these ITR sequences is within the skill in the art. (See, eg, Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520-532 (1996) text). An example of such a molecule employed in the present invention is a "cis-acting" plastid containing a transgene in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. Such AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the isolated nucleic acid additionally includes a region comprising a second AAV ITR (eg, a second region, a third region, a fourth region, etc.).

In addition to the major elements identified above for the recombinant AAV vector, the vector also includes well-known control elements to allow transcription, translation and/or expression of the transgene in cells transfected with the vector or infected with the virus produced by the invention means operably linked to elements of the transgene. As used herein, "operably linked" sequences include expression control sequences that are contiguous to the gene of interest and expression control sequences that act in trans or long-range to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyadenylation) signals; sequences that stabilize cytoplasmic mRNAs; sequences that enhance translation efficiency (eg, Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance secretion of the encoded product. A number of expression control sequences, including native, constitutive, inducible and/or tissue-specific promoters, are known in the art and available.

As used herein, a nucleic acid sequence (eg, a coding sequence) and a regulatory sequence are said to be operably linked when they are covalently linked in such a way that the expression or transcription of the nucleic acid sequence is placed under the influence or control of the regulatory sequence. connect. If the nucleic acid sequence is to be translated into a functional protein, if induction of the promoter in the 5' regulatory sequence results in transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) Two DNA sequences are considered operably linked if ) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to translate into protein. Thus, a promoter region will be operably linked to a nucleic acid sequence if the promoter region is capable of affecting the transcription of the DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide. Similarly, when two or more coding regions are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins that have been translated in frame, the two or more coding regions The divisions are operably linked. In some embodiments, operably linked coding sequences result in fusion proteins.

A region comprising a transgene (eg, comprising a fusion protein, etc.) can be located at any suitable location in an isolated nucleic acid that will express the fusion protein.

It will be appreciated that where the transgene encodes more than one polypeptide, each polypeptide may be located at any suitable position within the transgene. For example, the nucleic acid encoding the first polypeptide can be located in an intron of the transgene and the nucleic acid sequence encoding the second polypeptide can be located in another untranslated region (eg, in the last codon of the protein coding sequence as much as the transgene between the first bases of the adenylate signal).

"Promoter" refers to a DNA sequence recognized by a cell's synthetic machinery or introduced synthetic machinery and required to initiate specific transcription of a gene. The phrases "operably linked", "operably placed", "under control" or "under transcriptional control" mean that the promoter is in the correct position and or orientation relative to the nucleic acid to control RNA polymerase initiation and the expression of the gene.

For nucleic acids encoding proteins, the polyadenylation sequence is typically inserted after the transgene sequence and before the 3' AAV ITR sequence. The rAAV constructs used in the present invention may also contain introns, desirably located between the promoter/enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40 T intron sequence. Another vector element that can be used is the internal ribosome entry site (IRES). IRES sequences are used to generate more than one polypeptide from a single gene transcript. IRES sequences will be used to generate proteins containing more than one polypeptide chain. The selection of these and other common vector elements is known and many such sequences are available [see, eg, Sambrook et al., and references cited therein, for example, at pages 3.18-3.26 and 16.17-16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, the foot-and-mouth disease virus 2A sequence is included in a polyprotein; this is a small peptide (about 18 amino acids in length) that has been shown to mediate cleavage of polyproteins (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, N M et al, J Virology, Nov 1996; pp. 8124-8127; Furler, S et al, Gene Therapy, 2001; 8: 864-873; and Halpin, C et al Human, The Plant Journal, 1999; 4: 453-459). The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems including plastids and gene therapy vectors (AAV and retrovirus) (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, Nov 1996; pp. 8124-8127; Furler, S et al, Gene Therapy, 2001; 8: 864-873; and Halpin, C et al, The Plant Journal, 1999; 4: 453-459; de Felipe, P et al, Gene Therapy, 1999; 6: 198-208; de Felipe, P et al, Human Gene Therapy, 2000; 11: 1921-1931.; and Klump, H et al, Gene Therapy, 2001; 8: 811-817).

Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancers), the cytomegalovirus (CMV) promoter (optionally with CMV enhancer) [see, eg, Boshart et al., Cell, 41:521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, beta-actin promoter, phosphoglycerol kinase (PGK) promoter promoter and EF1α promoter [Invitrogen]. In some embodiments, the promoter is a P2 promoter. In some embodiments, the promoter is the chicken beta-actin (CBA) promoter. In some embodiments, the promoters are two CBA promoters. In some embodiments, the promoters are two CBA promoters separated by a CMV enhancer. In some embodiments, the promoter is the CAG promoter.

Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors (such as temperature), or the presence of a specific physiological state such as the acute phase, a specific differentiation state of a cell, or only in replicating cells ). Inducible promoters and inducible systems are available from various commercial sources including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and are readily selectable by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include zinc inducible ovine metallothionein (MT) promoter, dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, T7 Polymerase promoter system (WO 98/10088); ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), tetracycline repressible system (Gossen et al. , Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin Chem. Biol., 2:512-518 (1998)), RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432 -441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that can be used in the context are those that are regulated by a particular physiological state (eg, temperature, acute phase, a particular differentiation state of a cell, or only in replicating cells).

In another embodiment, the native promoter of the transgene will be used. The native promoter may be preferred when it is desired that the expression of the transgene should mimic native expression. The native promoter can be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In another embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic native expression.

In some embodiments, the regulatory sequences confer tissue-specific gene expression capabilities. In some instances, tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (eg, promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter , pancreatic polypeptide (PPY) promoter, synaptophysin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian ligamentin (DES) promoter, α-myosin heavy chain (α-MHC) ) promoter or the cardiac troponin T (cTnT) promoter. Other exemplary promoters include the beta-actin promoter, the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); the alpha-fetoprotein (AFP) promoter, Arbuthnot et al, Hum. Gene Ther., 7: 1503-14 (1996)), Osteocalcin promoter (Stein et al, Mol. Biol. Rep., 24: 185-96 (1997)); Bone sialoprotein Promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); Immunoglobulin Heavy chain promoters; T cell receptor alpha-chain promoters, neuronal (such as neuron-specific enolase (NSE) promoters) (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 ( 1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)) and neuron-specific vgf gene promoter (Piccioli et al., Neuron , 15:373-84 (1995)), among others, are known to the skilled artisan.

In some embodiments, a transgenic line encoding a fusion protein comprising DBD and a transactivator is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is neural tissue specific. In some embodiments, the promoter is a SST or NPY promoter.

Aspects of the invention relate to isolated nucleic acids comprising more than one promoter (eg, 2, 3, 4, 5 or more promoters). For example, in the context of a construct having a transgene comprising a first region encoding a protein and a second region encoding a protein, it may be desirable to use a first promoter sequence (eg, a first promoter operably linked to the protein encoding region) subsequence) drives the expression of the first protein-coding region, and a second promoter sequence (eg, a second promoter sequence operably linked to the second protein-coding region) drives the expression of the second protein-coding region. In general, the first promoter sequence and the second promoter sequence can be the same promoter sequence or different promoter sequences. In some embodiments, the first promoter sequence (eg, a promoter that drives expression of a protein coding region) is an RNA polymerase III (pol III) promoter sequence. Non-limiting examples of pol III promoter sequences include U6 and H1 promoter sequences. In some embodiments, the second promoter sequence (eg, the promoter sequence driving the second protein) is an RNA polymerase II (pol II) promoter sequence. Non-limiting examples of pol II promoter sequences include T7, T3, SP6, RSV and cytomegalovirus promoter sequences. In some embodiments, the pol III promoter sequence drives the expression of the first protein coding region. In some embodiments, the pol II promoter sequence drives the expression of the second protein coding region.

Recombinant Adeno-Associated Virus (rAAV) In some aspects, the present invention provides isolated adeno-associated virus (AAV). As used herein with respect to AAVs, the term "isolated" refers to AAVs that have been artificially produced or obtained. Isolated AAV can be produced using recombinant methods. These AVs are referred to herein as "recombinant AAVs." Recombinant AAV (rAAV) preferably has tissue-specific targeting capabilities such that the nuclease and/or transgene of the rAAV is specifically delivered to one or more predetermined tissues. The AAV capsid is an important element for determining these tissue-specific targeting capabilities. Therefore, rAAVs with capsids suitable for targeting tissues can be selected.

Methods for obtaining recombinant AAV with the desired capsid protein are well known in the art. (See eg, US 2003/0138772), the contents of which are incorporated herein by reference in their entirety). Typically these methods involve culturing host cells containing a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector consisting of an AAV inverted terminal repeat (ITR) and a transgene; and sufficient helper functions to allow The recombinant AAV vector is packaged within the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the cap gene of AAV. AAV contains three capsid proteins, virion proteins 1 to 3 (designated VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are about 87 kDa, about 72 kDa, and about 62 kDa, respectively. In some embodiments, upon translation, the capsid protein forms a spherical 60 monomer unit protein coat around the viral genome. In some embodiments, the function of the capsid proteins is to protect the viral genome, deliver the genome, and interact with the host. In some aspects, the capsid protein delivers the viral genome to the host in a tissue-specific manner.

In some embodiments, the AAV capsid protein has an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, and AAV.PHP. B. In some embodiments, the AAV capsid protein has a serotype derived from a non-human primate, eg, the AAVrh8 serotype. In some embodiments, the AAV capsid protein is of a serotype derived from a broad and efficient CNS transduction, eg, AAV.PHP.B. In some embodiments, the capsid protein is of AAV serotype 9.

Components to be cultured in the host cell to package the rAAV vector in the AAV capsid can be provided to the host cell in trans. Alternatively, any one or more of the desired components (eg, recombinant AAV vectors, rep sequences, cap sequences, and/or helper functions) can be provided by a stable host cell that has been Known methods are engineered to contain one or more of the desired components. Most suitably, this stable host cell will contain the desired components under the control of an inducible promoter. However, the one or more desired components may be under the control of a constitutive promoter. In discussing regulatory elements suitable for use with transgenes, examples of suitable inducible and constitutive promoters are provided herein. In yet another option, the selected stable host cell may contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, stable host cells can be generated that are derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contain rep and/or cap proteins under the control of an inducible promoter. Still other stable host cells can be generated by those skilled in the art.

In some embodiments, the invention pertains to host cells containing nucleic acid comprising a coding sequence encoding a transgene (eg, a DNA binding domain fused to a transcriptional regulatory domain). In some embodiments, the host cell is a mammalian cell, a yeast cell, a bacterial cell, an insect cell, a plant cell, or a fungal cell.

The recombinant AAV vectors, rep sequences, cap sequences and helper functions required to produce the rAAV of the invention can be delivered to the packaging host cell using any suitable genetic element (vector). The selected genetic elements can be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of the present invention are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Likewise, methods for producing rAAV virions are well known and the selection of a suitable method does not limit the present invention. See, eg, K. Fisher et al., J. Virol., 70:520-532 (1993) and US Patent No. 5,478,745.

In some embodiments, the triple transfection method (described in detail in US Pat. No. 6,001,650) can be used to generate recombinant AAV. Typically, recombinant AAV is produced by transfecting host cells with an AAV vector to be packaged in AAV particles (comprising a transgene flanked by ITR elements), an AAV helper function vector, and an accessory function vector. AAV helper vectors encode "AAV helper" sequences (eg, rep and cap sequences) that are used in trans for productive AAV replication and encapsidation. Preferably, the AAV helper vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (eg, AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present invention include pHLP19 described in US Patent No. 6,001,650 and the pRep6cap6 vector described in US Patent No. 6,156,303, both of which are incorporated herein by reference in their entirety middle. Accessory function vectors encode nucleotide sequences that are not AAV-derived viral and/or cellular functions that AAV relies on for replication (eg, "accessory functions"). Accessory functions include those required for AAV replication, including but not limited to those involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly . Virus-based accessory functions can be derived from any of known helper viruses such as adenovirus, herpes virus (except herpes simplex virus type 1), and vaccinia virus.

In some aspects, the invention provides transfected host cells. The term "transfection" is used to refer to the uptake of exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced into the interior of the cell membrane. A number of transfection techniques are generally known in the art. See, eg, Graham et al, (1973) Virology, 52:456, Sambrook et al, (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al, (1986) Basic Methods in Molecular Biology, Elsevier and Chu et al. (1981) Gene 13:197. These techniques can be used to introduce one or more exogenous nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, into suitable host cells.

"Host cell" refers to any cell that carries or is capable of carrying a substance of interest. Usually the host cell is a mammalian cell. In some embodiments, the host cell line neurons, optionally GABAergic neurons. As used herein, a "GABAergic neuron" is a nerve cell that produces gamma aminobutyric acid (GABA). In mammals, GABA is a neurotransmitter widely distributed in the nervous system, and GABA binds and inhibits the neurons to which it binds. As such, GABA has been implicated in many disorders affecting the nervous system, including epilepsy, autism, and anxiety. Studies in SCN1A hemizygous and knockout mice have observed severe sodium current deficits in GABAergic neurons in the brain. Host cells can be used as recipients for AAV helper constructs, AAV pocket gene plastids, accessory function vectors, or other transfer DNA associated with recombinant AAV production. The term includes progeny of the original cell that has been transfected. Thus, a "host cell" as used herein may refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell are not necessarily identical to the original parent in morphology or genome or total DNA complement due to natural, accidental or deliberate mutation.

As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Typically, a cell line is derived from a clonal population of single precursor cells. It is additionally known in the art that spontaneous or induced changes in the karyotype can occur during storage or transfer of these pure line populations. Thus, cells derived from a referenced cell line may not be identical to the previous cell or culture, and the referenced cell line includes such variants.

As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as RNA.

As used herein, the term "vector" includes any genetic element capable of replicating and transferring genetic sequences between cells, such as plastids, bacteriophages, transposons, cohesoplasts, chromosomes, artificial chromosomes, when combined with appropriate control elements , viruses, virions, etc. In some embodiments, the vector is a viral vector, such as an rAAV vector, lentiviral vector, adenoviral vector, retroviral vector, and the like. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, carefully considered useful vectors are those in which the nucleic acid segment to be transcribed is under the transcriptional control of a promoter.

"Promoter" refers to a DNA sequence recognized by a cell's synthetic machinery or introduced synthetic machinery and required to initiate specific transcription of a gene. The phrases "operably linked", "operably placed", "under control" or "under transcriptional control" mean that the promoter is in the correct position and or orientation relative to the nucleic acid to control RNA polymerase initiation and the expression of the gene. The term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of a nucleic acid coding sequence is capable of being transcribed. In some embodiments, expression includes transcription of nucleic acid, eg, to produce a biologically active polypeptide product from a transcribed gene. The foregoing methods for packaging the recombinant vector in the desired AAV capsid to generate the rAAV of the present invention are not intended to be limiting and other suitable methods will be known to the skilled artisan.

Methods for modulating target gene expression The present invention provides methods for modulating gene expression in cells or individuals. These methods generally involve administering to a cell or individual an isolated nucleic acid or rAAV comprising a transgene encoding a fusion protein comprising a DNA binding domain (eg, a ZFP domain) and a transactivation domain. In some embodiments, the fusion protein comprises a ZFP and a VP64 transactivator. In some embodiments, the fusion protein comprises a ZFP and a p65 transactivator. In some embodiments, the fusion protein comprises a ZFP and an RTA transactivator. In some embodiments, the fusion protein comprises a ZFP and a VPR transactivator. In some embodiments, the method involves administering to a cell or individual a dCas9 protein and at least one guide nucleic acid targeting SCN1A (e.g., comprising or consisting of any of SEQ ID NO: 83 to 94). guide nucleic acid encoded by any one of 94).

In some embodiments, administration of an isolated nucleic acid or rAAV encoding a fusion protein (eg, a fusion protein comprising a transactivator) to a cell or individual results in increased expression of a target gene (eg, SCN1A). Thus, in some embodiments, the compositions and methods described by the present invention are suitable for the treatment of disorders caused by haploinsufficiency of target genes, such as Zoffer syndrome caused by haploinsufficiency of the SCN1A gene.

As used herein, "haploinsufficiency" refers to where one copy of a gene (eg, SCN1A) is inactivated, eg, by genetic mutation or deletion, and the remaining functional copies of the gene are not sufficient to produce enough to maintain normal function of the gene A genetic disorder of the amount of the gene product.

Zhuofei Syndrome (also known as Myosic Infantile Severe Epilepsy) is a rare lifelong form of epilepsy that usually manifests within the first three years of life. Zhuofei syndrome is characterized by prolonged and frequent seizures, behavioral and developmental delays, motor and balance problems, language and speech delay problems, and disruption of the autonomic nervous system. In some embodiments, the individual has a haploinsufficiency associated with Zoffer syndrome, such as a mutation in one copy of the SCN1A gene, resulting in decreased SCN1A protein in the cell or individual. Most patients with Zoffer's syndrome carry SCN1A mutations that are translated into truncated proteins; other SCN1A mutations associated with Zoffer's syndrome include splice site and missense mutations, and are randomly distributed throughout the SCN1A gene mutation. In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the SCN1A gene. In some embodiments, compositions for targeting SCNA1 comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the SCN1A gene , gRNA).

In some embodiments, the individual has haploinsufficiency associated with MED13L haploinsufficiency syndrome, wherein the individual has only a single functional copy of the MED13L gene. Individuals with MED13L haploinsufficiency syndrome often have mutations in the second non-functional copy of their MED13L gene. MED13L haploinsufficiency syndrome is characterized by intellectual disability, speech problems, distinctive facial features, and developmental delay. In some embodiments, the fusion proteins of the present invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the MED13L gene. In some embodiments, compositions for targeting MED13L comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the MED13L gene. , gRNA).

In some embodiments, the individual has a haploinsufficiency associated with myelodysplastic syndrome. Individuals with myelodysplasia syndrome typically have mutations in one copy of the isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2) and/or GATA2 genes. Myelodysplastic syndromes are a group of cancers in which immature blood cells in the bone marrow do not mature into healthy blood cells. Sometimes, this syndrome can lead to acute myeloid leukemia. In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the IDH1 gene. In some embodiments, compositions for targeting IDH1 comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the IDH1 gene , gRNA). In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the IDH2 gene. In some embodiments, compositions for targeting IDH2 comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the IDH2 gene , gRNA). In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the GATA2 gene. In some embodiments, compositions for targeting GATA2 comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the GATA2 gene , gRNA).

In some embodiments, the individual has a haploinsufficiency associated with DiGeorge syndrome. Individuals with Diggeorg syndrome typically have deletions of 30 to 40 genes in the middle of chromosome 22 at a location called 22q11.2. Specifically, the disease may be characterized by haploinsufficiency of the TBX gene. Diggeorg syndrome is characterized by congenital heart disease, certain facial features, frequent infections, developmental delays, learning problems, and cleft palate. In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the TBX gene. In some embodiments, compositions for targeting TBX comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the TBX gene , gRNA).

In some embodiments, the individual has haploinsufficiency associated with CHARGE syndrome. In most cases, individuals with CHARGE syndrome are haploinsufficient in the CHD7 gene. CHARGE syndrome is characterized by eye defects, heart defects, posterior nasal atresia, growth and/or developmental delay, genital and/or urinary system abnormalities and ear abnormalities and deafness. In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the CHD7 gene. In some embodiments, compositions for targeting CHD7 comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the CHD7 gene , gRNA).

In some embodiments, the individual has haploinsufficiency associated with Ehlers-Danlos syndrome. Individuals with Ehlers-Danlos Syndrome may have haploinsufficiency of the following: COL1A1 , COL1A2 , COL3A1 , COL5A1 , COL5A2 , TNXB , ADAMTS2 , PLOD1 , B4GALT7 , DSE and/or D4ST1 / CHST14 genes. Ehlers-Danlos syndrome is characterized by hyperelastic skin and can lead to aortic dissection, scoliosis, and early-onset osteoarthritis. In some embodiments, fusion proteins of the invention comprise specifically targeting (eg, binding to) any of the COL1A1 , COL1A2 , COL3A1 , COL5A1 , COL5A2 , TNXB , ADAMTS2 , PLOD1 , B4GALT7 , DSE or D4ST1 / CHST14 genes The ZFP domain and the transactivation domain. In some embodiments, compositions for targeting any of COL1A1 , COL1A2 , COL3A1 , COL5A1 , COL5A2 , TNXB , ADAMTS2 , PLOD1 , B4GALT7 , DSE , or D4ST1 / CHST14 comprise (i) a dCas protein and a transfection Fusion proteins of activation domains, and (ii) specifically targeting (eg, binding to) any of the COL1A1 , COL1A2 , COL3A1 , COL5A1 , COL5A2 , TNXB , ADAMTS2 , PLOD1 , B4GALT7 , DSE , or D4ST1 / CHST14 genes Guide nucleic acid (eg, gRNA).

In some embodiments, the individual has haploinsufficiency associated with frontotemporal dementia (FTD). Individual lines with FTD were haploinsufficient in the MAPT gene (which encodes the Tau protein) and/or the GRN gene. FTD is characterized by memory loss, lack of social awareness, poor impulse control, and speech difficulties. In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the MAPT gene. In some embodiments, compositions for targeting MAPT comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the MAPT gene. , gRNA). In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) a GRN gene. In some embodiments, a composition for targeting GRN comprises (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the GRN gene , gRNA).

In some embodiments, the individual has a haploinsufficiency associated with Holt-Oram syndrome. Haploinsufficiency of the TBX5 gene in a system with Holt-Oram syndrome. Holt-Oram syndrome is characterized by cardiac complications, including congenital heart defects and cardiac conduction disorders. In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the TBX5 gene. In some embodiments, compositions for targeting TBX5 comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the TBX5 gene , gRNA).

In some embodiments, the individual has a haploinsufficiency associated with Marfan syndrome. Individuals with Marfan syndrome are usually haploinsufficient in the FBN1 gene, which encodes the fibrillin-1 protein. Marfan syndrome is characterized by disproportionate limb length, early-onset arthritis, cardiac complications, and/or dysfunction of the autonomic nervous system. In some embodiments, fusion proteins of the invention comprise a ZFP domain and a transactivation domain that specifically target (eg, bind to) the FBN1 gene. In some embodiments, compositions for targeting FBN1 comprise (i) a fusion protein comprising a dCas protein and a transactivation domain, and (ii) a guide nucleic acid (eg, binding to) that specifically targets (eg, binds to) the FBN1 gene , gRNA).

The present invention is based in part on methods of administering to an individual a fusion protein as described herein. In some embodiments, the fusion protein comprises DBD and a transcriptional activator. In some embodiments, the DBD is a ZNF, TALE, dCas protein (eg, dCas9 or dCas12a) or binds to the homeodomain of the SCN1A gene. In some embodiments, the transcriptional activator is VP64, p65, RTA or a triplex transcriptional activator comprising VP64-p65-RTA (VPR). In some embodiments, the fusion protein is flanked by AAV inverted terminal repeat (ITR) sequences. In some embodiments, the fusion protein is operably linked to a promoter. In some embodiments, the individual has or is suspected of having a mutation in SCN1A that results in a haploinsufficiency of the SCN1A protein. In some embodiments, the individual has or is suspected of having Zoffer syndrome.

In some aspects, the present invention provides methods of modulating (eg, increasing, decreasing, etc.) the expression of a target gene in a cell. In some embodiments, the present invention provides methods of increasing expression of a target gene (eg, SCN1A) in a cell. In some embodiments, the cell line is a mammalian cell. In some embodiments, the cell line is in an individual (eg, in vivo). In some embodiments, a systemic mammalian individual, such as a human. In some embodiments, the cell line is a nervous system cell (central nervous system cell or peripheral nervous system cell), such as a neuron (eg, GABAergic neuron, unipolar neuron, bipolar neuron, basket cell, Bayer cells, Lugaro cells, spiny neurons, Purkinje cells, pyramidal cells, Renshaw cells, granule cells, motor neurons, spindle cells, etc.) or Glial cells (eg, astrocytes, oligodendrocytes, ependymal cells, radial glial cells, Schwann cells, satellite cells, etc.).

In a "normal" cell or individual, the expression of a target gene (eg, SCN1A) is sufficient such that the cell or individual is not haploinsufficient for the target gene (eg, SCN1A). In some embodiments, the "improved" or "increased" expression or activity of a transgene is relative to the transgene in cells or individuals to which one or more isolated nucleic acids, rAAVs, or compositions as described herein have not been administered performance or activity. In some embodiments, the "improved" or "increased" performance or activity of a transgene is relative to the transgene at a time after the individual has been administered one or more isolated nucleic acids, rAAVs, or compositions as described herein. The expression or activity in the individual is measured (eg, gene expression is measured before and after administration of one or more isolated nucleic acids, rAAVs, or compositions as described herein). For example, in some embodiments, "improved" or "increased" expression of SCN1A in a cell or individual is measured relative to a cell or individual that has not been administered a transgene encoding a fusion ZFP-transactivator. In some embodiments, the methods described herein result in a 2- to 100-fold increase in SCN1A expression and/or activity in an individual relative to SCN1A expression and/or activity in an individual who has not been administered one or more of the compositions described herein (eg, 2x, 5x, 10x, 50x, 100x, etc.).

As used herein, the terms "treatment, treating" and "therapy" refer to both therapeutic treatment and prophylactic or preventative procedures. The term additionally includes amelioration of existing symptoms, prevention of additional symptoms, amelioration or prevention of underlying causes of symptoms, prevention or reversal of causes of symptoms, eg, symptoms associated with a haploinsufficiency gene (eg, haploinsufficiency SCN1A gene). Thus, the term refers to a favorable outcome conferred on an individual who has a disorder (eg, a disease or disorder associated with a haploinsufficiency gene, eg, Zoffer syndrome) or has the potential to develop such a disorder. In addition, the term "treating" also includes administering or administering an agent (eg, a therapeutic agent or a therapeutic combination) to an individual who may have a disease, a symptom of a disease, or a predisposition to a disease, or an isolated tissue or cell line from an individual. (e.g., an isolated nucleic acid or rAAV that targets or binds to a target gene or regulatory region of a target gene) for the purpose of curing, healing, alleviating, relieving, altering, remediating, ameliorating, ameliorating or affecting the disease, symptoms of the disease or have a tendency to suffer from disease.

A therapeutic agent or composition can include a compound in a pharmaceutically acceptable form that prevents and/or reduces the symptoms of a particular disease (eg, a disease or disorder associated with a haploinsufficiency gene, eg, Zoffer syndrome). For example, a therapeutic composition can be a pharmaceutical composition that prevents and/or reduces symptoms of a disease or disorder associated with a haploinsufficiency gene (eg, Zoffer syndrome). The therapeutic compositions of the present invention will be provided in any suitable form with due consideration. The form of the therapeutic composition will depend on many factors, including the mode of administration as described herein. The therapeutic composition may contain diluents, adjuvants and excipients and other ingredients as described herein.

investment mode The isolated nucleic acids, rAAVs, and compositions of the present invention can be delivered in compositions to an individual according to any suitable method known in the art. For example, an individual, ie, a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate, can be treated (eg, rhesus monkeys) rAAV is administered, preferably suspended in a physiologically compatible carrier (eg, in a composition). In some embodiments, the host animal does not include a human.

rAAV can be delivered to a mammalian subject, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the blood stream can be by injection into a vein, artery or any other vascular catheter. In some embodiments, the rAAV is administered into the bloodstream by means of isolated limb perfusion (a technique well known in the surgical field), which essentially allows the skilled artisan to circulate the limb to the system prior to administration of the rAAV virions separation. Skilled artisans may also employ a variant of the isolated limb perfusion technique described in US Pat. No. 6,177,403 to administer virions into the vasculature of isolated limbs to potentially enhance transduction into muscle cells or tissues. In addition, in certain instances, it may be desirable to deliver virions to the CNS of an individual. "CNS" means all cells and tissues of the brain and spinal cord of vertebrates. Thus, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), intercellular spaces, bone, cartilage, and the like. Recombinant AAV can be obtained using neurosurgical techniques known in the art such as by stereotaxic injection (see, eg, Stein et al, J Virol 73:3424-3429, 1999; Davidson et al, PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000) by injection into, for example, the ventricular area with a needle, catheter or related device Intra and injection into the striatum (eg, the caudate nucleus or putamen of the striatum), thalamus, spinal cord and neuromuscular junction, or cerebellar lobules for direct delivery to the CNS or brain. In some embodiments, the rAAV as described in the present invention is administered by intravenous injection. In some embodiments, the rAAV is administered by intracerebral injection. In some embodiments, the rAAV is administered by intrathecal injection. In some embodiments, the rAAV is administered by intrastriatal injection. In some embodiments, the rAAV is delivered by intracranial injection. In some embodiments, the rAAV is delivered by cisterna magna injection. In some embodiments, the rAAV is delivered by intraventricular injection.

Aspects of the invention pertain to compositions comprising recombinant AAV comprising a capsid protein and a nucleic acid encoding a transgene, wherein the transgene comprises nucleic acid sequences encoding one or more proteins. In some embodiments, the nucleic acid additionally comprises an AAV ITR. In some embodiments, the composition additionally comprises a pharmaceutically acceptable carrier.

Compositions of the invention may comprise rAAV alone, or in combination with one or more other viruses (eg, encoding a second rAAV with one or more different transgenes). In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each with one or more different transgenes.

Those skilled in the art can readily select an appropriate carrier in view of the indication for which the rAAV is directed. For example, one suitable carrier includes saline, which can be formulated with various buffered solutions (eg, phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, polydextrose, agar, pectin, peanut oil, sesame oil, and water. The choice of the carrier does not limit the invention.

Optionally, in addition to the rAAV and the carrier, the compositions of the present invention may also contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, p-chlorophenol, and polox Sham (non-ionic surfactant) such as Pluronic ^® F-68. Suitable chemical stabilizers include gelatin and albumin.

rAAV is administered in sufficient quantities to transfect cells of the desired tissue and provide sufficient quantities of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the organ of choice (eg, intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, Intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. Alternatively, routes of administration can be combined.

The dose of rAAV virions required to achieve a particular "therapeutic effect" (eg, dose units in gene copies per kilogram of body weight (GC/kg)) will vary based on several factors, including (but not limited to): The route of administration of the rAAV virions, the amount of gene or RNA expression required to achieve a therapeutic effect, the particular disease or disorder under treatment, and the stability of the gene or RNA product. Based on the foregoing factors and other factors well known in the art, one skilled in the art can readily determine a rAAV virion dosage range to treat a patient with a particular disease or disorder.

An effective amount of rAAV is an amount sufficient to target the infected animal, targeting the desired tissue. In some embodiments, an effective amount of rAAV is administered to the individual during the presymptomatic stage of the lysosomal storage disease. In some embodiments, the presymptomatic stage of the lysosomal storage disease occurs between birth (eg, perinatal period) and 4 weeks of age.

In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly in the presence of high rAAV concentrations (eg, -10 ¹³ GC/mL or greater). Methods for reducing rAAV aggregation are well known in the art and include, for example, the addition of surfactants, pH modifiers, salt concentration modifiers, and the like. (See eg, Wright FR et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference).

Those skilled in the art are familiar with the formulation of pharmaceutically acceptable excipients and carrier solutions, and are familiar with developing appropriate dosing and treatment regimens for use in various treatment regimens of the particular compositions described herein.

Typically, such formulations will contain at least about 0.1% active compound or more, although the percentage of active ingredient(s) may of course vary and may conveniently range from about 1 or 2% to about 1 or 2% by weight or volume of the total formulation Between 70% or 80% or more. The amount of active compound in each therapeutically useful composition can, of course, be prepared in such a way that an appropriate dosage will be obtained in any given unit dose of the compound. Those skilled in the preparation of such pharmaceutical formulations will carefully consider various factors (such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations) and, therefore, various dosages and treatment regimens may be suitable. required.

In certain instances, subcutaneous, intrapancreatic, intranasal, parenteral, intravenous, intramuscular, intrathecal or oral, intraperitoneal, or by inhalation delivery of a suitably formulated pharmaceutical composition based on Therapeutic constructs of rAAV may be desirable. In some embodiments, modes of administration as described in US Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each expressly incorporated herein by reference in its entirety) can be used to deliver rAAV. In some embodiments, the preferred mode of administration is by portal vein injection.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that it is easy to inject. This form must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, sorbic acid, sodium thiosalate, and the like. In many cases, it is preferred to include isotonic agents such as sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin in the compositions.

For administration of aqueous injectable solutions, for example, the solution may optionally be suitably buffered and the liquid diluent first isotonic with sufficient saline or dextrose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media known to those skilled in the art can be employed. For example, one dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous infusion solution or injected at the proposed infusion site (see, eg, "Remington's Pharmaceutical Sciences", 15th Edition, pp. 1035-1038 and 1570- 1580 pages). Depending on the condition of the host, some variation in dosage will necessarily occur. In any event, the responsible administrator will determine the appropriate dosage for the individual host.

Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in an appropriate solvent, optionally with various of the other ingredients enumerated herein, in an appropriate solvent followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof .

The rAAV compositions disclosed herein can also be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amine groups of proteins) and the like with inorganic acids (such as, for example, hydrochloric or phosphoric acid) or organic acids (such as acetic, oxalic, tartaric, mandelic acid) , and the like) are formed. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine , histidine, procaine and analogs). Once formulated, the solution will be administered in a manner compatible with the dosage formulation and in an amount that will render it therapeutically effective. These formulations are readily administered in a variety of dosage forms such as injectable solutions, drug release capsules, and the like.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions Liquids, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host.

The compositions of the present invention can be introduced into suitable host cells using delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like. In particular, transgenes delivered by rAAV vectors can be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres or nanoparticles or the like.

These formulations may preferably be used in pharmaceutically acceptable formulations incorporating the nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes is generally known to those skilled in the art. More recently, liposomes with improved serum stability and circulating half-life have been developed (US Pat. No. 5,741,516). In addition, various approaches have been described for liposomes and liposome-like formulations as potential drug carriers (US Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with many cell types that are generally resistant to transfection by other procedures. In addition, liposomes are not limited by the DNA length specific to virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, reflex therapeutics, viruses, transcription factors and allosteric effectors into various cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

Lipid systems are formed by dispersing phospholipids in an aqueous medium and spontaneously forming multilamellar concentric bilayer vesicles, also known as multilamellar vesicles (MLVs). MLVs typically have a diameter of 25 nm to 4 µm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å and containing aqueous solutions in their cores.

Alternatively, nanocapsule formulations of rAAV can be used. Nanocapsules can generally capture substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, these ultrafine particles (approximately 0.1 µm in size) should be designed using in vivo degradable polymers. The use of biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements is carefully considered.

In addition to the delivery methods described above, the following techniques are also carefully considered as alternative methods of delivering rAAV compositions to the host. Sonication (ie, ultrasound) has been used and described in US Pat. No. 5,656,016 as a device for enhancing the rate and effectiveness of drug penetration into and through the circulatory system. Other drug delivery alternatives that have been carefully considered are intraosseous injection (US Pat. No. 5,779,708), microchip devices (US Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (US Pat. No. 5,797,898), 5,770,219 and 5,783,208) and the delivery of feedback control (US Pat. No. 5,697,899).

example Example 1: Design of zinc finger proteins to upregulate SCN1A gene expression Regions of homology between human (HEK293T cells) and mouse (HEPG2 cells) SCN1A promoter sequences were identified by aligning sequences around two important transcription initiation sites identified in the RIKEN CAGE sequence dataset for each species (Fig. 1). A highly conserved sequence between human (HEK) and mouse (HEPG2) is present in the proximal promoter region of SCN1A (Figure 2). Three ZFPs consisting of six fingers were designed by assembling one- and two-finger modules with predetermined DNA binding specificities to bind overlapping 15-22 nucleotide homology regions in the SCN1A proximal promoter region (Figure 3). Three ZFPs (ZFP1 to ZFP3), each consisting of six fingers, were designed to combine the overlapping highly conserved sequences identified in Figure 3 . Each refers to a region of three bases (triplets) designed to bind to the highly conserved region of the SCN1A proximal promoter.

As shown in Figure 4A, ZFP-1 recognizes individual three-base regions within the proximal promoter region of the SCN1A gene (SEQ ID NO: 2) (DNA triplet shown in red, separated by "•"). As shown in Figure 4B, each recognition helix (seven amino acids) of fingers 1 to 6 of ZFP-1 binds three nucleotide sequences. The amino acid sequences of the six fingers of ZFP-1 are shown in Figure 4C (SEQ ID NOs: 17-22); the linkers between the fingers are highlighted to designate canonical (TGEKP) and atypical (TGSQKP) linker sequences. The nucleotide sequences of the six fingers of ZFP-1 are shown in Figure 4D (SEQ ID NOs: 11 to 16). Table 1: Recognition helix of ZFP-1 targeting SCN1A amino acid sequence Nucleotide sequence ZFP-1 recognizes helix 1 QRGNLVR (SEQ ID NO: 17) CAGCGGGGAAAACCTGGTGAGG (SEQ ID NO: 11) ZFP-1 recognizes helix 2 LSFNLTR (SEQ ID NO: 18) CTGAGCTTCAATCTAACCAGA (SEQ ID NO: 12) ZFP-1 recognizes helix 3 RSDNLTR (SEQ ID NO: 19) CGGAGTGACAACTTAACGCGG (SEQ ID NO: 13) ZFP-1 recognizes helix 4 DRSHLAR (SEQ ID NO: 20) GACCGGTCTCACCTTGCCCGA (SEQ ID NO: 14) ZFP-1 recognizes helix 5 QKAHLTA (SEQ ID NO: 21) CAGAAGGCCCATTTGACTGCC (SEQ ID NO: 15) ZFP-1 recognizes helix 6 RSDNLTR (SEQ ID NO: 22) CGGTCGGACAACCTCACACGC (SEQ ID NO: 16)

As shown in Figure 5A, ZFP-2 recognizes individual three-base regions within the proximal promoter region of the SCN1A gene (SEQ ID NO: 3) (DNA triplet represented in red, separated by "•"). As shown in Figure 5B, each recognition helix (seven amino acids) of fingers 1 to 6 of ZFP-2 binds three nucleotide sequences. The amino acid sequences of the six fingers of ZFP-2 are shown in Figure 5C (SEQ ID NOs: 29-34); the linkers between the fingers are highlighted to designate canonical (TGEKP) and atypical (TGSQKP) linker sequences. The nucleotide sequences of the six fingers of ZFP-1 are shown in Figure 5D (SEQ ID NOs: 23 to 28). Table 2: Recognition helix of ZFP-2 targeting SCN1A amino acid sequence Nucleotide sequence ZFP-2 recognizes helix 1 RSSNLTR (SEQ ID NO: 29) CGAAGTTCCAACCTGACACGG (SEQ ID NO: 23) ZFP-2 recognizes helix 2 DKRTLIR (SEQ ID NO: 30) GACAAGCGGACCTTAATCCGC (SEQ ID NO: 24) ZFP-2 recognizes helix 3 QRGNLVR (SEQ ID NO: 31) CAGCGGGGAAATCTAGTGCGA (SEQ ID NO: 25) ZFP-2 recognizes helix 4 LSFNLTR (SEQ ID NO: 32) CTGAGCTTCAACTTGACTCGT (SEQ ID NO: 26) ZFP-2 recognizes helix 5 RSDNLTR (SEQ ID NO: 33) CGGAGTGACAATCTTACGAGA (SEQ ID NO: 27) ZFP-2 recognizes helix 6 DRSHLAR (SEQ ID NO: 34) GACCGGAGCCACTTAGCCAGG (SEQ ID NO: 28)

As shown in Figure 6A, ZFP-3 recognizes individual three-base regions within the proximal promoter region of the SCN1A gene (SEQ ID NO: 4) (DNA triplet indicated in red, separated by "•"). As shown in Figure 6B, each recognition helix (seven amino acids) of fingers 1 to 6 of ZFP-3 binds three nucleotide sequences. The amino acid sequences of the six fingers of ZFP-3 are shown in Figure 6C (SEQ ID NOs: 41 to 46); the linkers between the fingers are highlighted to designate canonical (TGEKP) and atypical (TGSQKP) linker sequences. The nucleotide sequences of the six fingers of ZFP-1 are shown in Figure 6D (SEQ ID NOs: 35 to 40). Table 3: Recognition helix of ZFP-3 targeting SCN1A amino acid sequence Nucleotide sequence ZFP-3 recognizes helix 1 DRSALAR (SEQ ID NO: 41) GACCGGAGCGCGCTGGCACGG (SEQ ID NO: 35) ZFP-3 recognizes helix 2 RSDNLTR (SEQ ID NO: 42) CGAAGTGACAACTTAACGCGC (SEQ ID NO: 36) ZFP-3 recognizes helix 3 QSGDLTR (SEQ ID NO: 43) CAGTCAGGGGACCTCACTCGT (SEQ ID NO: 37) ZFP-3 recognizes helix 4 VRQTLKQ (SEQ ID NO: 44) GTACGACAGACGCTTAAACAA (SEQ ID NO: 38) ZFP-3 recognizes helix 5 AAGNLTR (SEQ ID NO: 45) GCCGCTGGTAACTTGACACGA (SEQ ID NO: 39) ZFP-3 recognizes helix 6 RSDNLTR (SEQ ID NO: 46) AGATCTGATAATCTAACGCGT (SEQ ID NO: 40)

Additional ZFPs designed to target conserved sequences in the proximal promoter region of the SCN1A gene will each contain five or six finger domains and will bind to 15 to 22 nucleosides that are highly conserved between human and mouse SCN1A Acid zone. Table 4: Zinc finger proteins targeting SCN1A amino acid sequence Nucleotide sequence ZFP-1 RPFQCRICMRNFSQRGNLVRHIRTHTGEKPFACDICGKKFALSFNLTRHTKIHTGSQKPFQCRICMRNFSRSDNLTRHIRTHTGEKPFACDICGKKFADRSHLARHTKIHTGSQKPFQCRICMRNFSQKAHLTAHIRTHTGEKPFACDICGRKFARSDNLTRHTKIHLRQKD (SEQ ID NO: 57) CGACCATTCCAGTGTCGAATCTGCATGCGCAACTTCAGCCAGCGGGGAAACCTGGTGAGGCATATCCGCACCCACACGGGAGAGAAGCCTTTTGCCTGCGATATTTGTGGAAAGAAGTTTGCTCTGAGCTTCAATCTAACCAGACACACCAAGATTCATACTGGGTCCCAGAAACCGTTCCAGTGTAGGATATGCATGAGGAATTTCTCTCGGAGTGACAACTTAACGCGGCATATAAGGACGCACACAGGTGAAAAACCATTTGCATGCGACATCTGTGGCAAAAAGTTTGCGGACCGGTCTCACCTTGCCCGACACACAAAAATCCATACCGGCAGTCAAAAGCCCTTTCAATGTCGCATTTGCATGCGAAACTTCTCACAGAAGGCCCATTTGACTGCCCATATTCGTACTCATACTGGCGAGAAACCTTTCGCTTGCGATATATGTGGTCGTAAGTTTGCACGGTCGGACAACCTCACACGCCACACTAAGATACACCTGCGGCAGAAGGAC (SEQ ID NO: 58) ZFP-2 RPFQCRICMRNFSRSSNLTRHIRTHTGEKPFACDICGKKFADKRTLIRHTKIHTGSQKPFQCRICMRNFSQRGNLVRHIRTHTGEKPFACDICGKKFALSFNLTRHTKIHTGSQKPFQCRICMRNFSRSDNLTRHIRTHTGEKPFACDICGRKFADRSHLARHTKIHLRQKD (SEQ ID NO: 59) CGACCATTCCAGTGTCGAATCTGCATGCGCAACTTCAGCCGAAGTTCCAACCTGACACGGCATATCCGCACCCACACGGGAGAGAAGCCTTTTGCCTGCGATATTTGTGGAAAGAAGTTTGCTGACAAGCGGACCTTAATCCGCCACACCAAGATTCATACTGGGTCCCAGAAACCGTTCCAGTGTAGGATATGCATGAGGAATTTCTCTCAGCGGGGAAATCTAGTGCGACATATAAGGACGCACACAGGTGAAAAACCATTTGCATGCGACATCTGTGGCAAAAAGTTTGCGCTGAGCTTCAACTTGACTCGTCACACAAAAATCCATACCGGCAGTCAAAAGCCCTTTCAATGTCGCATTTGCATGCGAAACTTCTCACGGAGTGACAATCTTACGAGACATATTCGTACTCATACTGGCGAGAAACCTTTCGCTTGCGATATATGTGGTCGTAAGTTTGCAGACCGGAGCCACTTAGCCAGGCACACTAAGATACACCTGCGGCAGAAGGAC (SEQ ID NO: 60) ZFP-3 RPFQCRICMRNFSDRSALARHIRTHTGEKPFACDICGKKFARSDNLTRHTKIHTGSQKPFQCRICMRNFSQSGDLTRHIRTHTGEKPFACDICGKKFAVRQTLKQHTKIHTGSQKPFQCRICMRNFSAAGNLTRHIRTHTGEKPFACDICGRKFARSDNLTRHTKIHLRQKD (SEQ ID NO: 61) CGACCATTCCAGTGTCGAATCTGCATGCGCAACTTCAGCGACCGGAGCGCGCTGGCACGGCATATCCGCACCCACACGGGAGAGAAGCCTTTTGCCTGCGATATTTGTGGAAAGAAGTTTGCTCGAAGTGACAACTTAACGCGCCACACCAAGATTCATACTGGGTCCCAGAAACCGTTCCAGTGTAGGATATGCATGAGGAATTTCTCTCAGTCAGGGGACCTCACTCGTCATATAAGGACGCACACAGGTGAAAAACCATTTGCATGCGACATCTGTGGCAAAAAGTTTGCGGTACGACAGACGCTTAAACAACACACAAAAATCCATACCGGCAGTCAAAAGCCCTTTCAATGTCGCATTTGCATGCGAAACTTCTCAGCCGCTGGTAACTTGACACGACATATTCGTACTCATACTGGCGAGAAACCTTTCGCTTGCGATATATGTGGTCGTAAGTTTGCAAGATCTGATAATCTAACGCGTCACACTAAGATACACCTGCGGCAGAAGGAC (SEQ ID NO: 62)

Example 2: ZFP increases SCN1A gene expression in human cells To examine the ability of ZFP1-ZFP3 to upregulate transcription of SCN1A, the ZFP1-ZFP3 DNA binding domain was fused to a hybrid VP64, p53 and RTA (VPR) triplet strong transcriptional activator domain to form a chimeric transactivator. VPR fusion activator domains serve to recruit transcriptional regulatory complexes and increase chromatin accessibility and help achieve high gene expression. Therefore, the ZFP domain would target the VPR activator to a highly conserved sequence in the proximal promoter region to increase SCN1A gene expression.

Expression plasmids encoding VPR-ZFP1, VPR-ZFP2 and/or VPR-ZFP3 fusion proteins were transfected into HEK293 cells via transient transfection and the SCN1A gene was measured by qRT-PCR (using TBP expression as a reference for normalization) Performance. VPR-ZFP fusions include ZFP1, ZFP2 and/or ZFP3 fused to VPR. Transfection of the three constructs for multiplex regulation (containing ZFP1, ZFP2 and ZFP3 DNA binding domains each fused to VPR) resulted in a 45-fold increase in SCN1A gene expression relative to untransfected cells, indicating VPR-ZFP chimerism Transactivators can increase SCN1A gene expression by binding in the proximal region of the gene promoter (Figure 7).

The VPR-[ZFP1-ZFP3] fusion protein and the VPR-ZFP fusion protein in which the ZFP DNA binding domain is currently being designed were transfected into HeLa and HEPG2 cells, both of which have low SCN1A expression. VPR-ZFP fusion proteins contain a combination of single and multiple ZFP DNA binding domains fused to VPR transactivators. SCN1A gene expression was measured by qRT-PCR to determine whether these VPR-ZFP fusions could increase gene expression. The most promising VPR-ZFP fusion candidates were tested for their ability to increase SCN1A expression in primary mouse cortical neurons following adeno-associated virus (AAV) delivery of these fusion proteins.

The specificity of the ZFP domain was further optimized using a bacterial single-hybrid selection system (see, eg, Meng et al., "Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases", Nat Biotechnol, 2008) to extract the most important role in DNA binding from the A random library of residue changes identifies ideal ZFPs. Newly selected ZFPs were fused to VPR transactivator domains individually and in multiple ZFP combinations and transfected in HEK293, HeLa and HEPG2 cells, and primary mouse cortical neurons for identification after qRT-PCR analysis Candidate ZFP domains that maximize the expression of the SCN1A gene.

Example 3: Generation of a series of ZFPs ^{SCN1A transactivators} with varying potency The most potent ZFPs expressed from the up-regulated SCN1A gene from Example 2 were fused to a series of human transactivation domains with expected potency gradients (eg, Rta, p65, Hsf1, etc.) , to identify assemblies that achieved a 2-fold upregulation of SCN1A gene expression across a range of AAV multiplicities of infection (MOI). Mouse primary cortical neurons from normal and SCN1A ^+/- mice were infected with AAV vectors expressing ZFP ^SCN1A fusion transactivators. The expression of Na _V 1.1 protein was assessed by Western blotting and using qPCR. Primary neurons treated with TGF-α for 8 hours were used as a positive control as this treatment increased Na _V 1.1 protein expression by -6- to 8-fold (Chen et al., 2015, Neuroinflammation 12: 126). Changes in expression levels of other _NaVα subunit genes were also assessed to confirm the specification of ZFP ^{SCN1A transactivation} . Immunofluorescence was used to determine whether Na _V 1.1 expression was still restricted to GABAergic interneurons, by using anti-ZFP ^SCN1A (HA tag) and markers specific for GABAergic neurons (e.g., microalbumin ⁺ or Double immunofluorescence staining was performed with antibodies to statin ⁺ ) or universal neuronal markers (eg, NeuN, TUBIII and/or Map2). The specificity of ZFP ^SCN1A for SCN1A gene transactivation was also assessed by ChIP-Seq and RNA-Seq to map gene body binding sites and the resulting transcriptomic profiles generated after gene transfer.

Example 4: Histone organization and epigenomic landscape of the SCN1A promoter in GABAergic inhibitors guides the design of promoter activity-dependent SCN1A-ZFP transactivators The ability of ZFPs to bind to genome targets depends on the availability of the target sequence and accessibility (eg, presence of nucleosome-free regions). This requirement of DNA accessibility was used to design ZFP transactivators that function only in a subset of cell types based on the presence of DNA target sequence accessibility. Additional restriction of cell type activity is achieved by using tissue-specific promoters expressed by ZFP transactivators. Small promoters from puffer fish (Takifugu rubripes) somatostatin and neuropeptide Y genes have been shown to drive high specificity in cortical and hippocampal inhibitory interneurons in the context of AAV vectors and lentiviruses Transgenic expression. In some embodiments, the combination of AAV-based transcriptional restriction of SCN1A-specific ZFPs sensitive to DNA accessibility results in highly specific upregulation of _NaV1.1 protein expression in inhibitory interneurons throughout the brain. This dual modulation approach minimizes the side effects that can result from ectopic expression of the Na _V 1.1 protein in cells that do not normally express it.

Analysis of the nucleosome structure and epigenetic landscape of the SCN1A promoter in mouse and human GABAergic inhibitory and glutamatergic excitatory neurons. This information was used to design GABAergic inhibitory neuron-restricted ZFP transactivators by targeting sequences accessible only around the SCN1A locus in this cell type.

GABAergic inhibitory neurons isolated from transgenic mice expressing TdTomato under the GAD67 promoter using fluorescence-activated cell sorting (FACS) and generated by crossing Emx1-IRES-Cre with ROSA26/stop/EGFP mice GFP-positive glutamatergic excitatory neurons. Human GABAergic and excitatory neurons were generated from induced pluripotent stem (iPS) cells and confirmed using immunostaining and RT-PCR, and electrophysiological activity using markers specific for these cell types. Accessible gene body regions around the SCN1A promoter in mouse and human neuronal populations were characterized using analysis of transposase accessible chromatin (ATAC-Seq).

Based on differential chromatin accessibility in the gene body regions surrounding the SCN1A promoter in inhibitory and excitatory neurons, ZFP ^SCN1A transactivators were designed that recognize sequences accessible only in GABAergic neurons. A series of candidate ZFP-VPR transactivator fusions were generated to target different SCN1A accessible regions, where binding of the transactivators is expected to efficiently upregulate _NaV 1.1 expression in inhibitory regions and reveal _NaV in excitatory neurons 1.1 Any undesired induced performance of the performance.

Performance studies were performed in cultured human iPS-derived neurons and mouse SCN1A ^+/- primary neurons mimicking Zoffer's syndrome to identify ZFP ^SCN1A transducers designed to identify DNA sequences accessible only in inhibitory neurons Whether activators provide the necessary specificity when expressed from AAV vectors under pan-neuronal human synaptophysin-1 or inhibitory interneuron-specific promoters. By qRT-PCR, Western blotting and inhibitory GABAergic (eg, GABA ⁺ , GAD65/67 ⁺ , somatostatin and/or microalbumin) and excitatory glutaminergic (eg, Cux1+, FoxG1+, Dual immunofluorescence measurements of neuron type-specific markers of GABA _A receptors, GABA ^- neurons) of Na _V 1.1 expression. The cell-type specificity of the ZFP ^{SCN1A transactivator} was designed to target different sequences in the mouse and human SCN1A promoters because the chromatin structure and DNA sequence within the syntenic region vary by species. Controls in these experiments included neuronal cultures infected with similar AAV vectors encoding GFP, ZFP without the transactivation domain, or transactivator without the ZFP DNA binding domain.

MicroRNA (miRNA) binding sites are incorporated into the 3' untranslated region (3' UTR) of ZFP ^{SCN1A transactivators} that are restricted to cell types in which undesired performance occurs (e.g., glutamatergic excitatory neurons). This approach was previously used to limit the expression of transgenes delivered by AAV (Xie et al., "MicroRNA-regulated, systematically delivered rAAV9: a step closer to CNS-restricted transgene expression", Mol. Ther. 2011). Differences in miRNA expression profiles in GABAergic inhibitory neurons and other cell types were determined by small RNA sequencing.

Example 5: Assessing the potential of AAV-ZFP ^SCN1A gene therapy to correct sodium current deficits in patient-derived iPS-producing GABAergic interneurons Key steps in the development of ZFP ^{SCN1A transactivators} for Zoffer syndrome were demonstrated These artificial transactivators have desired functions in human neurons. For this purpose, iPS cells from Zhuofei patients (n=4 to 6) and non- Zhuofei patients (n=4) were obtained. These cells display a non-Zuofei genetic background without the need for manual gene expression, and thus iPS cells have become the current state-of-the-art cell line for biomedical research. Isogenic cell lines were generated using CRISPR-Cas9 genome editing technology by repairing genetic mutations in SCN1A to wild-type sequences, or by introducing Zhuofei-related mutations into normal counterpart genes in control cell lines. Isogenic lines thereby eliminate the natural variability that arises from comparing cell lines from different human individuals, and are therefore valuable for confirming and increasing disease-specific phenotypes. Established inhibitory neuron differentiation protocols and validated pathways were used to differentiate iPS cell lines into forebrain GABAergic inhibitor interneurons.

Inhibitory neurons derived from Zhuo Fei patients showed reduced sodium current and impaired action potential firing as determined by whole-cell clamp electrophysiological measurements. Similar measurements were performed to confirm that the Zhuo Fei-derived neurons described herein reproduce the phenotypes associated with these diseases. Defects in sodium current occur in inhibitors, but not in excitatory neurons in Zhuo Fei patients (Sun et al.) and therefore only inhibitory neurons are used in the present invention. Mutation-induced defects in sodium channels in inhibitory neurons derived from Zhuo Fei patients were rescued by ectopic expression of wild-type SCN1A (ref. 20). Therefore, in the context of Zhuofei syndrome, the methods described in the present invention are suitable for testing the efficacy of ZFP ^{SCN1A transactivator} in restoring wild-type sodium channel function and physiology.

GABAergic inhibitory neuronal cultures were infected with AAV vectors encoding the ZFP ^{SCN1A transactivator} under either a universal neuron or inhibitory neuron specific promoter. Changes in the expression of Na _V 1.1 were assessed by Western blotting. Restoration of functional sodium currents in inhibitory neurons was assessed by whole cell clamping in untransfected cells compared to transfected cells. Binding of the ZFP ^{SCN1A transactivator} in the gene body in inhibitory neurons from all patients was analyzed by ChIP-seq and correlated with any identified transcriptomic changes detected by RNA-seq. Controls in these experiments were neuronal cultures infected with similar AAV vectors encoding GFP, ZFP without the VPR transactivator domain, and VPR transactivator without the ZFP DNA binding domain.

Example 6: Assessing the therapeutic potential of AAV-ZFP ^SCN1A intervention in SCN1A mice at different ages and routes of delivery The broad tropism of AAV is a key property for gene therapy applications of widely expressed genes, but when concerns about transgenic When expressed in a cell-type-specific manner, this can become a major challenge. This problem is largely addressed by using tissue-specific promoters such as thyroxine-binding protein (TBP), creatine kinase and troponin T, respectively, for major tissues in the body such as liver, muscle and heart. Additional control levels can be superimposed on tissue-specific promoters by incorporating multiple binding sites for microRNAs that are highly abundant in those tissues, such as miR-122 in liver and miR-1 in skeletal muscle Copies achieve a higher degree of off-target from specific tissues. The recently described AAV-PHP.B serotype, which induces a wide range of cell types, is exceptionally efficient for CNS gene transfer following systemic delivery. Furthermore, the tropism of this AAV-PHP.B serotype to peripheral tissues is largely as broad as that of AAV9. The aim of the gene therapy approach for Zoffe syndrome is to restore Na _V 1.1 expression only in GABAergic inhibitory interneurons, while preventing the deleterious effects of ectopic expression in other neurons and elsewhere. AAV and lentiviral vectors encoding GFP under small promoters (<2.8 kb) derived from the somatostatin (fSST) and neuropeptide Y (fNPY) genes of the puffer fish (Fugu puffer fish) have been shown to be free after intracranial injection. Driver inhibitory neuron-specific expression in the mouse brain. AAV-PHP.B vectors carrying these GFP expression-driving promoters were compared to control vectors, in which transgene expression is driven by the ubiquitous strong CAG promoter and the minimal relatively weak mouse MeCP2 promoter. Delivery to the CNS by systemic administration in 6-week-old (tail vein) and postnatal day 1 (retro-orbital) mice, CSF delivery in neonates, and finally unilateral injection targeting the dentate gyrus (DG) Next, the specificity of the AAV-PHP.B-GFP vector with the fSST and fNYP promoters for GABAergic inhibitory interneurons was investigated (Table 5). The efficiency of CNS gene transfer varies significantly by route of delivery and since Scn1a ^+/- mice of different ages were treated, extensive analyses were performed to establish the effect of each delivery route on neuronal transduction of GABAergic inhibitory interneurons throughout the CNS and promoter-specific baselines. AAV vectors driving GFP expression from short fSST and fNYP promoters have previously been shown to be highly specific for inhibitory interneurons in the hippocampus after direct injection. The AAV-PHP.B vector of the present invention was validated in the same manner as in a follow-up study in which it was assessed to restore inhibitory neurons (specifically located in the dentate gyrus and inner layer of the granulosa cell layer) in hippocampal formation in Scn1a ^+/- mice. Therapeutic effects of Na _V 1.1 manifestations in (middle) (the rationale is explained below). Experiments were performed in UMMS-generated 129SvJ/C57BL/6 mice by mating 129SvJ with C57BL/6 mice obtained from Jackson Laboratories (Bar Harbor, ME). Mice were euthanized one month after injection and brains and spinal cords were harvested for histological analysis of transduction efficiency and specificity using double immunofluorescence using antibodies against cell-specific markers and GFP. Gene transfer efficiency of GABAergic inhibitory interneurons was assessed in the whole brain and spinal cord by double immunofluorescence staining with antibodies to glutamic acid decarboxylase (GAD; a marker of GABAergic neurons) and GFP and specificity. In addition, the promoter and/or AAV-PHP.B were assessed for their effects on epistatin (SST), microalbumin (PV), calbindin (CR), vasoactivity using antibodies specific for these proteins and GFP Preferential specificity of a subset of inhibitory interneurons for gut peptide (VIP) or neuropeptide Y (NPY). Liver, heart, and skeletal muscle were collected from mice treated by systemic and ICV administration to assess GFP expression histologically and Western blotting was used to determine the possibility of ectopic expression in peripheral tissues. Table 5: Experimental groups Number of mice per cohort delivery route whole body ICV IC age 6 weeks* PND1 ^# PND1 ^# 8 weeks* Dose (vg) 2x10 ¹² 4x10 ¹¹ 4x10 ¹⁰ 1x10 ¹⁰ AAV-fSST-GFP 6 6-8 6-8 4 AAV-fNYP-GFP 6 6-8 6-8 4 AAV-CAG-GFP 6 6-8 6-8 4 AAV-MeCP2-GFP 6 6-8 6-8 - vehicle (PBS) 2 - - ^* Groups consisted of equal numbers of mice from both sexes. #Inject one litter per vehicle. Abbreviations: ICV: intraventricular injection; IC: intracranial injection; PND1: postnatal day 1

AAV-PHP.B vectors encoding different ZFP ^{Scn1a transactivator} proteins or the same volume of phosphate buffered saline (PBS) (n=3 males + 3 females/group) were administered bilaterally to six-week-old Scn1a In the dentate gyrus of ^+/- mice, the AAV-PHP.B vector is a construct with the ZFP ^Scn1a activating domain but without the effects of the DNA binding domain controlling the activator alone. The single-stranded AAV vector used in these experiments also carried the IRES-GFP cassette downstream of the ZFP ^Scn1a cDNA to facilitate identification of transduced cells. At least two ZFP ^{Scn1a transactivators} were tested that could have broader activation in various neurons, as well as the two most promising GABAergic inhibitory neuron-restricted ZFP ^Scn1A transactivators described above. One month after injection, the brains were harvested and the hippocampus from one cerebral hemisphere was dissected to assess ZFP ^Scn1a , Na _V 1.1, Na _V 1.3, GAD65, GAD65, β-actin or tubulin as loading controls by Western blotting. Expression of GAD67 protein. The other cerebral hemisphere was examined by histological studies using serial brain sections (10 µm) by using antibodies against GAD and GFP, or anti-GAD and epitope tags (HA or myc tags) included in all ZFP ^Scn1a proteins. Double immunofluorescence staining of antibodies against ) to analyze the % of transduced inhibitory interneurons in the dentate gyrus and lobules within the granulosa cell layer. Likewise, the percentage of GAD-positive neurons expressing Na _V 1.1 and Na _V 1.3 was determined to demonstrate restoration of the normal pattern of sodium channel expression. In addition to the immunofluorescence detection of Na _V 1.1 and Na _V 1.3 protein expression, RNAscope probes for Na _V 1.1, Na _V 1.3, ZFP ^Scn1a and GAD were used to assess changes in mRNA levels in GABAergic interneurons. RNAScope is a highly sensitive in situ hybridization technique to analyze the amount of mRNA in brain neurons. The combination of these two methods to assess changes in _NaV1.1 amounts caused by ZFP ^Scn1a expression provides a comprehensive understanding of how interneuron changes are achieved by the gene therapy methods of the present invention.

The therapeutic efficacy of AAV-PHP.B-ZFP ^Scn1a gene therapy was analyzed in Scn1a ^+/- mice of both sexes starting at postnatal day 1 or 6 weeks of age via the tail vein. Controls included mice treated with an AAV vector encoding a ZFP-like protein without the ZFP DNA binding domain, and age-matched untreated Scn1a ^+/- mice and wild-type littermates (n=15 males per group) and 15 females). A subset of mice in each group (n=3 males and 3 females) were euthanized at 12 weeks of age using Western blotting and the use of anti-GAD (and other neuron type-specific markers, e.g., GAD65, GAD67) The efficiency of gene transfer to GABAergic interneurons and the restoration of Na _V 1.1 expression in these cells throughout the brain and spinal cord were assessed by immunofluorescence with antibodies to ZFP. In addition, ectopic manifestations of ZFPs and Na _V 1.1 manifestations in peripheral tissues were assessed. An additional subset of animals in each group (n=24) were used to study effects on survival (up to one year of age), motor performance and behavior, and were tested every two months from 2 to 12 months of age. Motor function and coordination were assessed using accelerated rotarod and beam tests, as Scn1a ^+/- mice showed impaired coordination of forelimbs and hindlimbs by PND21. In addition, severe impairment in Scn1a ^+/- mice was tested using behavioral tests in which Scn1a ^+/- mice showed impaired performance, including: open field, elevated plus maze, nesting, marble burial, and Barnes maze spatial learning and memory. The characteristics of spontaneous seizures in patients with Zhuofei syndrome were also evident in Scn1a ^+/- mice, and the frequency increased with age and body temperature. Furthermore, Scn1a ^+/- mice experienced premature sudden death immediately after tonic-clonic seizures. Therefore, 24-hour continuous video monitoring was used to assess seizure frequency and duration at 2, 6, and 12 months of age. If significant changes were detected in the primary outcomes measured in the tests described above, a social interaction study using chamber preference readouts to novel objects, odors, and mouse responses was considered. Brain, spinal cord, and peripheral organs were collected and assessed for molecular and histological analyses outlined above at the time of humane endpoint experiments.

Example 7: ZFP and dCas9 systems increase SCN1A gene expression in human cells To examine the ability of ZFP1-ZFP3 to upregulate transcription of SCN1A, the ZFP1-ZFP3 DNA binding domain was fused to a hybrid VP64, p53 and RTA (VPR) triplet strong transcriptional activator domain to form a chimeric transactivator. VPR fusion activator domains serve to recruit transcriptional regulatory complexes and increase chromatin accessibility and help achieve high gene expression. Therefore, the ZFP domain would target the VPR activator to a highly conserved sequence in the proximal promoter region to increase SCN1A gene expression.

In addition, to examine the ability of the dCas9 system targeting SCN1A to upregulate transcription of SCN1A, three guide RNAs targeting SCN1A were complexed with the dCas9 protein.

HEK293T cells were transiently transfected with any of the following experimental conditions: (1) VPR-ZFP1 construct; (2) VPR-ZFP2 construct; (3) VPR-ZFP3 construct; (4) VPR-ZFP1, VPR - All three of ZFP2 and VPR-ZFP3 constructs; (5) dCas9-VPR construct and SCN1A guide RNA 1; (6) dCas9-VPR construct and SCN1A guide RNA 2; (7) dCas9-VPR construct and SCN1A guide RNA 3; (8) dCas9-VPR construct and all three of SCN1A guide RNA 1, SCN1A guide RNA 2 and SCN1A guide RNA 3; and (9) dCas9-VPR construct without any guide RNA ( control). SCN1A gene expression was measured by qRT-PCR. Fold activation of SCN1A was normalized to control experiments (dCas9-VPR construct without any guide RNA).

All tested experimental conditions resulted in increased gene activation of SCN1A relative to control experiments (Figure 8). These data demonstrate that the zinc finger proteins described in this example and throughout this invention are capable of targeting SCN1A to affect gene expression. These data additionally demonstrate that the guide RNA sequences of this example (SEQ ID NOs: 83-94) are capable of targeting dCas9 to SCN1A to affect gene expression. Table 6: Guide nucleic acids targeting SCN1A (spacer sequences shown in bold) Nucleotide sequence (DNA) Nucleotide sequence (RNA) SCN1A Boot 1 GAGGTACCATAGAGTGAGGCG GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 83) GAGGUACCAUAGAGUGAGGCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 84) SCN1A Boot 2 ACCGAGGCGAGGATGAAGCCGAG GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 87) ACCGAGGCGAGGAUGAAGCCGAG GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 88) SCN1A bootstrap 3 ACCGAAGCCGAGAGGATACTGCAG GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 91) ACCGAAGCCGAGAGGAUACUGCAG GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 92)

Example 8: SCN1A-specific zinc finger protein (ZFP) transactivators with wide-ranging potencies A zinc finger protein (ZFP) transactivator (eg, with 2 fingers) targeting the conserved region of the SCN1A promoter was developed using the bacterial monohybrid (B1H) system for ZFP assembly. A B1H 2-finger ZFP module (2FM) library was then generated for each 6 base pair subsite within the larger ZFP recognition sequence and B1H selection was performed to identify candidate 2FMs for each 6 bp subsite. In addition, the DNA binding specificity of 2FMs was assessed to select 2FMs with preferential specificity for each of the 6 base pair subsites within the target recognition sequence.

Each combination of the characterized and selected 2-finger modules (2FM) was then assembled into a 6-finger ZFP with improved DNA binding specificity. These 6-finger ZFPs were expressed and purified in vitro and then assessed for DNA binding specificity using SELEX-seq and/or CUT&Tag in HEK293T cells. 6-finger ZFPs that target SCN1A with preferential specificity for >14 bases within each target site were identified such that at least one 6-finger ZFP was identified for three different target sequences.

The affinity of the 6-finger ZFPs was then fine-tuned to improve genome-wide binding profiles and the effect of inter-finger linker changes on genome-wide binding profiles was assessed (eg, using CUT&Tag). In addition, the effect of reduced non-specific DNA binding affinity on genome-wide binding profiles was assessed (eg, using CUT&Tag in HEK293T cells). Candidate 6 finger ZFPs were additionally assessed (eg, using SELEX-seq or CUT&Tag) for DNA binding specificity. These studies identified at least three different ZFPs targeting SCN1A with improved distribution of binding sites within the gene.

ZFP-transactivator fusion proteins were then identified that target SCN1A with varying potency in neurons. Candidate activation domain (AD) modules (eg, 8 to 12 different human activation domains (AD) from transcription factors present in GABAergic neurons) are evaluated in neurons. Candidate ADs were fused to lead candidate ZFPs and subsequent fusion proteins were assessed for their ability to activate SCN1A in HEK293T cells relative to synthetic ADs with established potency such as VP16, VP64 and VPR. qRT-PCR data were used to verify the functionality and activation potential of the ZFP _Scn1A -AD fusion protein.

The activation profile of ZFP _Scn1A -AD in iCell GABAergic inhibitory neurons was assessed. Each major ZFP _Scn1A -AD was transduced into iCell GABAergic inhibitory neurons and assessed for effects on SCN1A performance (eg, using qRT-PCR and Western blotting) and transcriptome (eg, using RNA-seq). qRT-PCR and RNA-seq data allow quantitative analysis (eg, volcano plots) of the effect of each AD on SCN1A expression and transcriptome homeostasis.

Evaluation of ZFP _Scn1A -AD candidates based on genome-wide binding maps in neurons. Each candidate ZFP _Scn1A -AD was transduced into iCell GABAergic inhibitory neurons and assessed (eg, using CUT&Tag) for DNA binding specificity and genome-wide profiles. Effects on SCN1A performance (eg, using qRT-PCR and Western blotting) and transcriptome (eg, using RNA-seq) were assessed. Off-target sites (eg, unwanted gene activation) are identified using these measurements.

The specificity of ZFP _Scn1A -AD for SCN1A binding (vs. off-target binding) was adjusted using a B1H counter-selection strategy to identify ZFP fingers that differentiate between on-target and off-target sites. The DNA binding specificity and genome-wide profiles of the candidate ZFP _Scn1A -AD were reassessed. SCN1A activation of the best ADs associated with the revised ZFP _Scn1A -AD was reassessed to confirm the selective activation of SCN1A by each candidate ZFP _Scn1A -AD.

The lead candidate ZFP _Scn1A -AD was evaluated in the wild-type mouse central nervous system (CNS). Wild-type mice (4 to 5 weeks old) were systemically treated with ^2x1012 vg AAV-PHP.eB vector encoding candidate ZFP _Scn1a -AD with various activation potentials. Controls included wild-type mice treated with AAV vectors encoding ZFPs without the DNA binding domain, and untreated mouse controls. Each ZFP _Scn1a -AD treatment group consisted of 2 male mice and 2 female mice. Outcome measures in life were survival, standard behavioral assessments, and standard health assessments. Animals were euthanized 5 weeks after treatment for molecular and histological analysis. Autopsy results were assessed by western blotting and histological analysis of SCN1A and ZFP _Scn1a protein levels, and snRNA-seq analysis of transcriptomic changes in transduced and non-transduced cells.

The binding profile of the potent AAV-ZFP _Scn1a vector in mouse experiments was then assessed, and the binding profile of the AAV-ZFP _Scn1a vector in the mouse CNS and SCN1A activation were assessed. The Cre driver and loxP-nGFP mice were used to selectively label different subsets of GABAergic neurons. Mice (4 to 5 weeks old) were treated systemically with 2x10 ¹² vg of AAV-PHP.eB vector encoding the candidate ZFP _Scn1a -AD. Controls included wild-type mice treated with AAV vectors encoding ZFPs without the DNA binding domain, and untreated mouse controls. Each ZFP _Scn1a -AD treatment group consisted of 2 male mice and 2 female mice. Outcome measures in life were survival, standard behavioral assessments, and standard health assessments. Animals were euthanized 5 weeks after treatment for molecular and histological analysis. Autopsy outcomes were assessed by FACS analysis of GABAergic neuron nuclei and transcriptome analysis using single nucleus RNAseq (snRNAseq). ZFP genome-wide binding assays were assessed by CUT&Tag. The most potent AAV-ZFP _Scn1a vector was then selected based on its ability to activate Scn1a with minimal impact on neuronal transcriptomic profiles. The therapeutic utility of the most promising carriers with a range of activation potential can then be assessed.

Example 9: GABAergic Neuron-Specific Gene Expression System Transgenic expression cassettes specific for inhibitory GABAergic interneurons were developed using three methods as described below.

Method 1 : Bioinformatics-guided design of GABA -specific promoters Figure 10, method 1 is depicted in the left panel.

Genome-wide ATACseq, ChIPseq, Dnase I, CAGE and HiC datasets from mouse and human brain were analyzed to find candidate enhancer elements for use in SCN1A, GAD1 and GAD2 promoters. HiC data for human Chr.4 near SCN1A show a 3D intrachromosomal interacting region that may indicate enhancers (~1 Mb centered on SCN1A; arrows indicate potential interactions between different regions of chromosome 2 165 to 166 Mb apart ). Interchromosomal interactions were also assessed.

Raw data can be generated using the Cre driver mouse line for a subset of GABA neurons (Scn1a, Gad2, Sst, Cck, Vip promoters) crossed with loxP-GFP mice (Figure 10, left panel, shaded box). GABA neurons were sorted and the epigenetic landscape of Scn1a, Gad1, Gad2 genes was assessed using CUT&Tag, and chromosomal interactions specific for GABA neurons were also assessed using HiC.

Bioinformatics-generated enhancer candidates for GABA neuron-specific performance were evaluated. A barcode library of enhancer candidates fused to the SCN1A minimal promoter was generated.

10 ¹² vg AAV-PHP.eB pool was systemically infused into 6- to 8-week-old normal mice (n=4) with experimental endpoints at 4 to 6 weeks post-injection. Outcome measures were snRNAseq expressing barcode distribution in CNS cell populations; RT-PCR amplification of barcodes in liver, heart and skeletal muscle followed by NGS analysis of frequency. Unique gene expression profiles were used to identify distinct cell populations in the analyzed tissues. Enhancers were selected for further study based on their ability to equal specific driver gene expression (unique barcodes) in GABA neurons, and a second selection criterion was to identify GABA-specific enhancers with varying degrees of potency. Three to four enhancers can be selected for comparison with those identified in Method 2.

Method 2 : Long-range enhancer sub-scan arrays generate AAV capsid libraries with > ^4x109 variants. These capsid libraries were used to probe for the presence of tissue-specific enhancers throughout the gene body region. A pool of -98,800 oligonucleotides (140 nucleotides in length) was synthesized and cloned upstream of the minimal SCN1A promoter in the AAV-nlsGFP vector. The oligonucleotides are tiled over the entire gene body region (SCN1A, GAD1, GAD2 genes in mouse and human) and the offset between oligonucleotides (one nucleotide to no overlap) determines the target Sizes ranged in size from ~99 kb to 13 Mb gene body regions. This AAV library carries 18 nucleotide barcodes (NNM6 = ~ ¹⁰⁹ barcodes), which are unique to each enhancer, located in the 3'UTR of the transgenic mRNA. The barcodes associated with each enhancer were determined by NGS after removal of the nlsGFP cDNA using low frequency restriction enzymes. The number of barcode reads in GFP-positive nuclei of the CNS and peripheral tissues provides a measure of specificity and overall transgene expression efficiency. Using the AAV Enhancer Scanning Array (AAVeSA) library allows rapid development of cell type specific enhancers for any cell type by sampling the uniquely expressed genes in the target.

Oligonucleotide pools of human and mouse SCN1A, GAD1 and GAD2 genes with 20 nucleotide offsets were generated to probe the ~2Mb region surrounding each gene. Array libraries were scanned using AAV enhancers with a minimum 100x coverage of all sequences (3 genes x 2 species x 98,800 oligonucleotides/gene = 592,800 sequences), or 5.9x107 variants. The identifiers of all enhancer barcodes (theoretically ~100 barcodes/enhancer) were determined by NGS as described above prior to generation.

The AAVeSA library was packaged in AAV-PHP.eB for systemic delivery.

In vivo screening of GABA neuron-specific enhancers was performed by systemic infusion of 10 ¹² vg AAVeSA pools in 6- to 8-week-old normal mice (n=4) with endpoints at 4-6 weeks post-injection. Outcome measures were snRNAseq expressing barcode distribution in CNS cell populations; RTPCR amplification of barcodes in liver, heart, skeletal muscle followed by NGS analysis of frequency. Enhancers were selected for further studies based on their specificity to GABA neurons in the CNS and covering a range of gene expression levels.

The GABA-specific AAV vectors were compared to each other. The AAV-nlsGFP vector carrying the enhancers selected above was packaged with AAV-PHP.eB and studied separately. Three to four vectors from each method were selected for further study.

10 ¹² vg AAV-PHP.eB-nlsGFP vector was systemically infused into 6- to 8-week-old normal mice (n=4/vector), and the experimental endpoint was 4 to 6 weeks post-injection. Outcome measures are GFP and neuronal (NeuN) cell-specific markers of GABAergic neurons (GAD1, and other markers of subsets of GABA neurons), astrocytes (ALDH1L1), and microglia (Iba1). Double immunofluorescence staining of brain sections; Western blot analysis of GFP expression in liver, heart, skeletal muscle; vector gene biodistribution in brain and the same peripheral organs. The expected result is the definition of a set of enhancers (combinations of enhancers) that provide varying amounts of gene expression restricted to GABAergic neurons.

Approach 3 : Post- transcriptional miR off- targets of gene expression from non- GABA neuronal cell populations in the CNS Existing data on miR expression profiles in mouse brain have been analyzed and a number of candidates have been selected that should be eliminated from the brain GABA neurons and most neuronal populations outside of astrocytes and microglia express off-target genes. An oligonucleotide library was generated covering a large number of combinations of miR targets (miR-T) and a large number of repeats to be cloned in the 3'UTR of an AAV vector expressing nlsGFP under the human synaptophysin-1 promoter. All miR-T cassettes carry the targets of miR-1 and miR-122 to express off-target genes from muscle and liver, respectively. The reason for including this feature is to allow the use of more ubiquitous promoters of varying strengths to drive ZFP expression. The performance of Scn1a can be fine-tuned by altering promoter strength and ZFP potency. Finally, each miR-T combination was associated with a unique barcode for RNAseq analysis. The library approach allows rapid selection of the most efficient combination of elements for GABA neuron-specific performance, which can be combined with other enhancers identified in Method 2.

The AAV-Syn1-nlsGFP-miRT library was generated by designing and constructing the library for cloning into the 3'UTR of the transgenic cassette. The NGS analysis uses the plastid library of the principles described above.

Generated using the AAV-PHP.eB library.

In vivo screening of GABA neuron-specific miR-T cassettes using systemic infusion of 10 ¹² vg AAV.miR-T library in 6- to 8-week-old normal mice (n=4) with endpoints 4 to 6 post-injection week. Outcome measures were snRNAseq expressing barcode distribution in CNS cell populations; RTPCR amplification of barcodes in liver, heart, skeletal muscle followed by NGS analysis of frequency.

The miR-T cassette was validated to restrict transgene expression to GABA neurons in the CNS. The top miR-T cassette was tested alone in the transgenic cassette encoding nlsGFP driven by the Syn-1 and CBA promoters. The AAV-PHP.eB-nlsGFP vector [(2 miR-T cassettes + no miR-T) x 2 promoters = 6 vectors) was studied alone.

10 ¹² vg AAV-PHP.eB-nlsGFP vector was systemically infused into 6- to 8-week-old normal mice (n=4/vector), and the experimental endpoint was 4 to 6 weeks post-injection. Outcome assessments are based on GFP and neuron (NeuN) cell-specific markers of GABAergic neurons (GAD1, other markers of a subset of GABA neurons), astrocytes (ALDH1L1), and microglia (Iba1). Double immunofluorescence staining of brain sections; Western blot analysis of GFP expression in liver, heart, skeletal muscle; vector gene biodistribution in brain and same peripheral organs. The expected outcome is the definition of one or more miR-T boxes that can restrict gene expression to GABAergic neurons.

The final AAV vector design carrying the selected enhancer and miR-T cassette was selected. Three GABA-specific enhancers covering a range of potencies were combined with the top miR-T cassette and studied individually - a total of at least six AAV vector designs.

10 ¹² vg AAV-PHP.eB-nlsGFP vector (6 vector design) was systemically infused into 6- to 8-week-old normal mice (n=4/vector), and the experimental endpoint was 4 to 6 weeks after injection. Outcome measures were snRNAseq; GFP and neuron (NeuN) cell specificity of GABAergic neurons (GAD1, and other markers of subsets of GABA neurons), astrocytes (ALDH1L1), microglia (Iba1) Double immunofluorescence staining of brain sections for markers; Western blot analysis of GFP expression in liver, heart, skeletal muscle; vector gene biodistribution in brain and same peripheral organs. The expected outcome was to select at least three AAV vectors with varying potency in most GABAergic neuron populations and no transgene expression in the CNS or other cell types of peripheral tissues.

Figure 1 shows the indicated human (HEK) and mouse (HEPG2) SCN1A genes (consensus sequence - SEQ ID NO: 98; target sequence - SEQ ID NO: 99; Hep-SCN1A_R4 sequence (top) - SEQ ID NO: 100; Hep - Chromatographic sequencing data on sequence conservation between SCN1A_R4 sequences (bottom) - SEQ ID NO: 101).

Figure 2 shows a sequence alignment of the proximal promoter regions of the human (SEQ ID NO: 1) and mouse (SEQ ID NO: 2) SCN1A genes, with the conserved sequences highlighted. Within this conserved sequence is the target region of interest for the zinc finger protein (ZFP) binding region, which is shown in bold (SEQ ID NO: 4).

Figure 3 shows the location of three overlapping target ZFP (ZFP-1, ZFP-2, ZFP-3) (SEQ ID NO: 5 to 7) binding sites in the proximal promoter region of the SCN1A gene (SEQ ID NO. : 3) schematic diagram.

Figures 4A to 4D show an alignment of the six recognition helical sequences of individual zinc fingers (finger 1 to finger 6; F1 to F6) in ZFP-1 that will recognize the SCN1A gene (SEQ ID NO: 2) Individual three-base regions within the proximal promoter region (DNA triplet shown in red, separated by "•"). Figure 4A highlights the nucleotide sequence (SEQ ID NO: 3) to which zinc fingers 1 to 6 (F1 to F6) of ZFP-1 will bind. Figure 4B shows the three nucleotide sequences recognized by each of the recognition helices (seven amino acids) of fingers 1 to 6 (SEQ ID NOs: 17 to 22) of ZFP-1. Figure 4C shows the amino acid sequence of ZFP-1 containing 6 fingers, one in each row, with the linkers between the fingers highlighted to designate the canonical (TGEKP) and atypical (TGSQKP) linker sequences (SEQ ID NO: 65 to 70). Figure 4D shows the nucleotide sequences (SEQ ID NOs: 102 to 107) of ZFP-1 (F1 to F6).

Figures 5A-5D show an alignment of the six recognition helical sequences of individual zinc fingers (finger 1 to finger 6; F1 to F6) in ZFP-2 that will recognize the SCN1A gene (SEQ ID NO: 3). Individual three base regions within the proximal promoter region (DNA triplets represented in red, separated by "*"). Figure 5A highlights the nucleotide sequence (SEQ ID NO: 3) to which zinc fingers 1 to 6 (F1 to F6) of ZFP-2 will bind. Figure 5B shows the first three nucleotides recognized by each of the recognition helices (seven amino acids) of fingers 1 to 6 (SEQ ID NOs: 29 to 34) of ZFP-2. Figure 5C shows the amino acid sequence of ZFP-2, which contains 6 fingers, one per row (SEQ ID NOs: 69 to 74), wherein the linkers between the fingers are highlighted to designate typical (TGEKP) and SARS type (TGSQKP) linker sequence. Figure 5D shows the nucleotide sequences (SEQ ID NOs: 108 to 113) of ZFP-2 (F1 to F6).

Figures 6A-6D show an alignment of the six recognition helical sequences of individual zinc fingers (finger 1 to finger 6; F1 to F6) in ZFP-3 that will recognize the SCN1A gene (SEQ ID NO: 4). Individual three base regions within the proximal promoter region (DNA triplets represented in red, separated by "*"). Figure 6A highlights the nucleotide sequence (SEQ ID NO: 3) to which zinc fingers 1 to 6 (F1 to F6) of ZFP-3 will bind. Figure 6B shows the first three nucleotides recognized by each of the recognition helices (seven amino acids) of fingers 1 to 6 (SEQ ID NOs: 41 to 46) of ZFP-3. Figure 6C shows the amino acid sequence of ZFP-3 containing 6 fingers, one per row (SEQ ID NOs: 75 to 80), wherein the linkers between the fingers are highlighted to designate typical (TGEKP) and SARS type (TGSQKP) linker sequence. Figure 6D shows the nucleotide sequences (SEQ ID NOs: 114 to 119) of ZFP-3 (F1 to F6).

Figure 7 shows data indicating that the SCN1A-binding ZFPs described in Figures 4-6 increased SCN1A gene expression in HEK293T cells as measured by quantitative real-time polymerase chain reaction (qRT-PCR). These expression constructs were delivered to cells by transient transfection of expression plastids encoding the following transcriptional regulators: Streptococcus pyogene Cas9 + SCN1A guide RNA (SpCas9 + Scn1a); Cas9 without endonuclease activity ( dCas9); VPR activation domain + SCN1A guide RNA (dCas9_VPR + Scn1a); VPR activation domain + ZFP1 (VPR_ZFP1); VPR activation domain + ZPF2 (VPR_ZFP2); VPR activation domain + ZFP3 (VPR_ZFP3); SpCas9 + ASCL1 guide RNA (SpCas9 + Ascl1); three VPR_ZFPs (VPR_ZFP1 + VPR_ZFP2 + VPR_ZFP3). Expression levels were normalized to TBP expression levels determined by qRT-PCR in each sample.

Figure 8 shows data indicating that the SCN1A-binding ZFP and Cas9+SCN1A guide RNA described in Figures 4-6 increased SCN1A gene expression in HEK293T cells, as measured by quantitative real-time polymerase chain reaction (qRT-PCR).

Figure 9 shows high-throughput chromosome conformation capture (Hi-C) data in the ~1 Mb range centered on SCN1A. Arrows indicate potential interactions between different regions of chromosome 2 separated by 165 to 166 Mb.

Figure 10 shows three methods for developing GABA-neuron-specific AAV vectors.

                                 
           <![CDATA[ <110> University of Massachusetts]]>
           <![CDATA[ <120> DNA binding domain transactivators and their uses]]>
           <![CDATA[ <130> U0120.70147WO00]]>
           <![CDATA[ <140>TW 110127164]]>
           <![CDATA[ <141> 2017-07-23]]>
           <![CDATA[ <150> US 63/056,528]]>
           <![CDATA[ <151> 2020-07-24]]>
           <![CDATA[ <160> 140 ]]>
           <![CDATA[ <170> PatentIn version 3.5]]>
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          cgaagttcca acctgacacg g 21
           <![CDATA[ <210> 24]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 24]]>
          gacaagcgga ccttaatccg c 21
           <![CDATA[ <210> 25]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 25]]>
          cagcgggggaa atctagtgcg a 21
           <![CDATA[ <210> 26]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 26]]>
          ctgagcttca acttgactcg t 21
           <![CDATA[ <210> 27]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 27]]>
          cggagtgaca atcttacgag a 21
           <![CDATA[ <210> 28]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 28]]>
          gaccggagcc acttagccag g 21
           <![CDATA[ <210> 29]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 29]]>
          Arg Ser Ser Asn Leu Thr Arg
          1 5
           <![CDATA[ <210> 30]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 30]]>
          Asp Lys Arg Thr Leu Ile Arg
          1 5
           <![CDATA[ <210> 31]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 31]]>
          Gln Arg Gly Asn Leu Val Arg
          1 5
           <![CDATA[ <210> 32]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 32]]>
          Leu Ser Phe Asn Leu Thr Arg
          1 5
           <![CDATA[ <210> 33]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 33]]>
          Arg Ser Asp Asn Leu Thr Arg
          1 5
           <![CDATA[ <210> 34]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 34]]>
          Asp Arg Ser His Leu Ala Arg
          1 5
           <![CDATA[ <210> 35]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 35]]>
          gaccggagcg cgctggcacg g 21
           <![CDATA[ <210> 36]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 36]]>
          cgaagtgaca acttaacgcg c 21
           <![CDATA[ <210> 37]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 37]]>
          cagtcagggg acctcactcg t 21
           <![CDATA[ <210> 38]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 38]]>
          gtacgacaga cgcttaaaca a 21
           <![CDATA[ <210> 39]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 39]]>
          gccgctggta acttgacacg a 21
           <![CDATA[ <210> 40]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 40]]>
          agatctgata atctaacgcg t 21
           <![CDATA[ <210> 41]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 41]]>
          Asp Arg Ser Ala Leu Ala Arg
          1 5
           <![CDATA[ <210> 42]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 42]]>
          Arg Ser Asp Asn Leu Thr Arg
          1 5
           <![CDATA[ <210> 43]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 43]]>
          Gln Ser Gly Asp Leu Thr Arg
          1 5
           <![CDATA[ <210> 44]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 44]]>
          Val Arg Gln Thr Leu Lys Gln
          1 5
           <![CDATA[ <210> 45]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 45]]>
          Ala Ala Gly Asn Leu Thr Arg
          1 5
           <![CDATA[ <210> 46]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 46]]>
          Arg Ser Asp Asn Leu Thr Arg
          1 5
           <![CDATA[ <210> 47]]>
           <![CDATA[ <211> 1569]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 47]]>
          gaggccagcg gttccggacg ggctgacgca ttggacgatt ttgatctgga tatgctggga 60
          agtgacgccc tcgatgattt tgaccttgac atgcttggtt cggatgccct tgatgacttt 120
          gacctcgaca tgctcggcag tgacgccctt gatgatttcg acctggacat gctgattaac 180
          tctagaagtt ccggatctag ccagtacctg cccgacaccg acgaccggca ccggatcgag 240
          gaaaagcgga agcggaccta cgagacattc aagagcatca tgaagaagtc ccccttcagc 300
          ggccccaccg accctagacc tccacctaga agaatcgccg tgcccagcag atccagcgcc 360
          agcgtgccaa aacctgcccc ccagccttac cccttcacca gcagcctgag caccatcaac 420
          tacgacgagt tccctaccat ggtgttcccc agcggccaga tctctcaggc ctctgctctg 480
          gctccagccc ctcctcaggt gctgcctcag gctcctgctc ctgcaccagc tccagccatg 540
          gtgtctgcac tggctcaggc accagcaccc gtgcctgtgc tggctcctgg acctccacag 600
          gctgtggctc caccagcccc taaacctaca caggccggcg agggcacact gtctgaagct 660
          ctgctgcagc tgcagttcga cgacgaggat ctgggagccc tgctgggaaa cagcaccgat 720
          cctgccgtgt tcaccgacct ggccagcgtg gacaacagcg agttccagca gctgctgaac 780
          cagggcatcc ctgtggcccc tcacaccacc gagcccatgc tgatggaata ccccgaggcc 840
          atcacccggc tcgtgacagg cgctcagagg cctcctgatc cagctcctgc ccctctggga 900
          gcaccaggcc tgcctaatgg actgctgtct ggcgacgagg acttcagctc tatcgccgat 960
          atggatttct cagccttgct gggctctggc agcggcagcc gggattccag ggaagggatg 1020
          ttttttgccga agcctgaggc cggctccgct attagtgacg tgtttgaggg ccgcgaggtg 1080
          tgccagccaa aacgaatccg gccatttcat cctccaggaa gtccatgggc caaccgccca 1140
          ctccccgcca gcctcgcacc aacaccaacc ggtccagtac atgagccagt cgggtcactg 1200
          accccggcac cagtccctca gccactggat ccagcgcccg cagtgactcc cgaggccagt 1260
          cacctgttgg aggatcccga tgaagagacg agccaggctg tcaaagccct tcgggagatg 1320
          gccgatactg tgattcccca gaaggaagag gctgcaatct gtggccaaat ggacctttcc 1380
          catccgcccc caaggggcca tctggatgag ctgacaacca cacttgagtc catgaccgag 1440
          gatctgaacc tggactcacc cctgaccccg gaattgaacg agattctgga taccttcctg 1500
          aacgacgagt gcctcttgca tgccatgcat atcagcacag gactgtccat cttcgacaca 1560
          tctctgttt 1569
           <![CDATA[ <210> 48]]>
           <![CDATA[ <211> 523]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 48]]>
          Glu Ala Ser Gly Ser Gly Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu
          1 5 10 15
          Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu
                      20 25 30
          Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp
                  35 40 45
          Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Ile Asn Ser Arg Ser Ser
              50 55 60
          Gly Ser Ser Gln Tyr Leu Pro Asp Thr Asp Asp Arg His Arg Ile Glu
          65 70 75 80
          Glu Lys Arg Lys Arg Thr Tyr Glu Thr Phe Lys Ser Ile Met Lys Lys
                          85 90 95
          Ser Pro Phe Ser Gly Pro Thr Asp Pro Arg Pro Pro Pro Arg Arg Ile
                      100 105 110
          Ala Val Pro Ser Arg Ser Ser Ala Ser Val Pro Lys Pro Ala Pro Gln
                  115 120 125
          Pro Tyr Pro Phe Thr Ser Ser Leu Ser Thr Ile Asn Tyr Asp Glu Phe
              130 135 140
          Pro Thr Met Val Phe Pro Ser Gly Gln Ile Ser Gln Ala Ser Ala Leu
          145 150 155 160
          Ala Pro Ala Pro Pro Gln Val Leu Pro Gln Ala Pro Ala Pro Ala Pro
                          165 170 175
          Ala Pro Ala Met Val Ser Ala Leu Ala Gln Ala Pro Ala Pro Val Pro
                      180 185 190
          Val Leu Ala Pro Gly Pro Pro Gln Ala Val Ala Pro Pro Ala Pro Lys
                  195 200 205
          Pro Thr Gln Ala Gly Glu Gly Thr Leu Ser Glu Ala Leu Leu Gln Leu
              210 215 220
          Gln Phe Asp Asp Glu Asp Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp
          225 230 235 240
          Pro Ala Val Phe Thr Asp Leu Ala Ser Val Asp Asn Ser Glu Phe Gln
                          245 250 255
          Gln Leu Leu Asn Gln Gly Ile Pro Val Ala Pro His Thr Thr Glu Pro
                      260 265 270
          Met Leu Met Glu Tyr Pro Glu Ala Ile Thr Arg Leu Val Thr Gly Ala
                  275 280 285
          Gln Arg Pro Pro Asp Pro Ala Pro Ala Pro Leu Gly Ala Pro Gly Leu
              290 295 300
          Pro Asn Gly Leu Leu Ser Gly Asp Glu Asp Phe Ser Ser Ile Ala Asp
          305 310 315 320
          Met Asp Phe Ser Ala Leu Leu Gly Ser Gly Ser Gly Ser Arg Asp Ser
                          325 330 335
          Arg Glu Gly Met Phe Leu Pro Lys Pro Glu Ala Gly Ser Ala Ile Ser
                      340 345 350
          Asp Val Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg Ile Arg Pro
                  355 360 365
          Phe His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro Leu Pro Ala Ser
              370 375 380
          Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu Pro Val Gly Ser Leu
          385 390 395 400
          Thr Pro Ala Pro Val Pro Gln Pro Leu Asp Pro Ala Pro Ala Val Thr
                          405 410 415
          Pro Glu Ala Ser His Leu Leu Glu Asp Pro Asp Glu Glu Thr Ser Gln
                      420 425 430
          Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro Gln Lys
                  435 440 445
          Glu Glu Ala Ala Ile Cys Gly Gln Met Asp Leu Ser His Pro Pro Pro
              450 455 460
          Arg Gly His Leu Asp Glu Leu Thr Thr Thr Leu Glu Ser Met Thr Glu
          465 470 475 480
          Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu Asn Glu Ile Leu
                          485 490 495
          Asp Thr Phe Leu Asn Asp Glu Cys Leu Leu His Ala Met His Ile Ser
                      500 505 510
          Thr Gly Leu Ser Ile Phe Asp Thr Ser Leu Phe
                  515 520
           <![CDATA[ <210> 49]]>
           <![CDATA[ <211> 6027]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 49]]>
          atggaacaga ccgtgctggt gccgccgggc ccggatagct ttaacttttt tacccgcgaa 60
          agcctggcgg cgattgaacg ccgcattgcg gaagaaaaag cgaaaaaccc gaaaccggat 120
          aaaaaagatg atgatgaaaa cggcccgaaa ccgaacagcg atctggaagc gggcaaaaac 180
          ctgccgttta tttatggcga tattccgccg gaaatggtga gcgaaccgct ggaagatctg 240
          gatccgtatt atattaacaa aaaaaccttt attgtgctga acaaaggcaa agcgattttt 300
          cgctttagcg cgaccagcgc gctgtatatt ctgaccccgt ttaacccgct gcgcaaaatt 360
          gcgattaaaa ttctggtgca tagcctgttt agcatgctga ttatgtgcac cattctgacc 420
          aactgcgtgt ttatgaccat gagcaacccg ccggattgga ccaaaaacgt ggaatatacc 480
          tttaccggca tttatacctt tgaaagcctg attaaaatta ttgcgcgcgg cttttgcctg 540
          gaagatttta cctttctgcg cgatccgtgg aactggctgg attttaccgt gattaccttt 600
          gcgtatgtga ccgaatttgt ggatctgggc aacgtgagcg cgctgcgcac ctttcgcgtg 660
          ctgcgcgcgc tgaaaaccat tagcgtgatt ccgggcctga aaaccattgt gggcgcgctg 720
          attcagagcg tgaaaaaact gagcgatgtg atgattctga ccgtgttttg cctgagcgtg 780
          tttgcgctga ttggcctgca gctgtttatg ggcaacctgc gcaacaaatg cattcagtgg 840
          ccgccgacca acgcgagcct ggaagaacat agcattgaaa aaaacattac cgtgaactat 900
          aacggcaccc tgattaacga aaccgtgttt gaatttgatt ggaaaagcta tattcaggat 960
          agccgctatc attattttct ggaaggcttt ctggatgcgc tgctgtgcgg caacagcagc 1020
          gatgcgggcc agtgcccgga aggctatatg tgcgtgaaag cgggccgcaa cccgaactat 1080
          ggctatacca gctttgatac ctttagctgg gcgtttctga gcctgtttcg cctgatgacc 1140
          caggattttt gggaaaacct gtatcagctg accctgcgcg cggcgggcaa aacctatatg 1200
          atttttttttg tgctggtgat ttttctgggc agcttttatc tgattaacct gattctggcg 1260
          gtggtggcga tggcgtatga agaacagaac caggcgaccc tggaagaagc ggaacagaaa 1320
          gaagcggaat ttcagcagat gattgaacag ctgaaaaaac agcaggaagc ggcgcagcag 1380
          gcggcgaccg cgaccgcgag cgaacatagc cgcgaaccga gcgcggcggg ccgcctgagc 1440
          gatagcagca gcgaagcgag caaactgagc agcaaaagcg cgaaagaacg ccgcaaccgc 1500
          cgcaaaaaac gcaaacagaa agaacagagc ggcggcgaag aaaaagatga agatgaattt 1560
          cagaaaagcg aaagcgaaga tagcattcgc cgcaaaggct ttcgctttag cattgaaggc 1620
          aaccgcctga cctatgaaaa acgctatagc agcccgcatc agagcctgct gagcattcgc 1680
          ggcagcctgt ttagcccgcg ccgcaacagc cgcaccagcc tgtttagctt tcgcggccgc 1740
          gcgaaagatg tgggcagcga aaacgatttt gcggatgatg aacatagcac ctttgaagat 1800
          aacgaaagcc gccgcgatag cctgtttgtg ccgcgccgcc atggcgaacg ccgcaacagc 1860
          aacctgagcc agaccagccg cagcagccgc atgctggcgg tgtttccggc gaacggcaaa 1920
          atgcatagca ccgtggattg caacggcgtg gtgagcctgg tgggcggccc gagcgtgccg 1980
          accagcccgg tgggccagct gctgccggaa gtgattattg ataaaccggc gaccgatgat 2040
          aacggcacca ccaccgaaac cgaaatgcgc aaacgccgca gcagcagctt tcatgtgagc 2100
          atggattttc tggaagatcc gagccagcgc cagcgcgcga tgagcattgc gagcattctg 2160
          accaacaccg tggaagaact ggaagaaagc cgccagaaat gcccgccgtg ctggtataaa 2220
          tttagcaaca tttttctgat ttgggattgc agcccgtatt ggctgaaagt gaaacatgtg 2280
          gtgaacctgg tggtgatgga tccgtttgtg gatctggcga ttaccatttg cattgtgctg 2340
          aacaccctgt ttatggcgat ggaacattat ccgatgaccg atcattttaa caacgtgctg 2400
          accgtgggca acctggtgtt taccggcatt tttaccgcgg aaatgtttct gaaaattatt 2460
          gcgatggatc cgtattatta ttttcaggaa ggctggaaca tttttgatgg ctttattgtg 2520
          accctgagcc tggtggaact gggcctggcg aacgtggaag gcctgagcgt gctgcgcagc 2580
          tttcgcctgc tgcgcgtgtt taaactggcg aaaagctggc cgaccctgaa catgctgatt 2640
          aaaattattg gcaacagcgt gggcgcgctg ggcaacctga ccctggtgct ggcgattatt 2700
          gtgtttattt ttgcggtggt gggcatgcag ctgtttggca aaagctataa agattgcgtg 2760
          tgcaaaattg cgagcgattg ccagctgccg cgctggcata tgaacgattt ttttcatagc 2820
          tttctgattg tgtttcgcgt gctgtgcggc gaatggattg aaaccatgtg ggattgcatg 2880
          gaagtggcgg gccaggcgat gtgcctgacc gtgtttatga tggtgatggt gattggcaac 2940
          ctggtggtgc tgaacctgtt tctggcgctg ctgctgagca gctttagcgc ggataacctg 3000
          gcggcgaccg atgatgataa cgaaatgaac aacctgcaga ttgcggtgga tcgcatgcat 3060
          aaaggcgtgg cgtatgtgaa acgcaaaatt tatgaattta ttcagcagag ctttattcgc 3120
          aaacagaaaa ttctggatga aattaaaccg ctggatgatc tgaacaacaa aaaagatagc 3180
          tgcatgagca accataccgc ggaaattggc aaagatctgg attatctgaa agatgtgaac 3240
          ggcaccacca gcggcattgg caccggcagc agcgtggaaa aatatattat tgatgaaagc 3300
          gattatatga gctttattaa caacccgagc ctgaccgtga ccgtgccgat tgcggtgggc 3360
          gaaagcgatt ttgaaaacct gaacaccgaa gattttagca gcgaaagcga tctggaagaa 3420
          agcaaagaaa aactgaacga aagcagcagc agcagcgaag gcagcaccgt ggatattggc 3480
          gcgccggtgg aagaacagcc ggtggtggaa ccggaagaaa ccctggaacc ggaagcgtgc 3540
          tttaccgaag gctgcgtgca gcgctttaaa tgctgccaga ttaacgtgga agaaggccgc 3600
          ggcaaacagt ggtggaacct gcgccgcacc tgctttcgca ttgtggaaca taactggttt 3660
          gaaaccttta ttgtgtttat gattctgctg agcagcggcg cgctggcgtt tgaagatatt 3720
          tatattgatc agcgcaaaac cattaaaacc atgctggaat atgcggataa agtgtttacc 3780
          tatattttta ttctggaaat gctgctgaaa tgggtggcgt atggctatca gacctatttt 3840
          accaacgcgt ggtgctggct ggattttctg attgtggatg tgagcctggt gagcctgacc 3900
          gcgaacgcgc tgggctatag cgaactgggc gcgattaaaa gcctgcgcac cctgcgcgcg 3960
          ctgcgcccgc tgcgcgcgct gagccgcttt gaaggcatgc gcgtggtggt gaacgcgctg 4020
          ctgggcgcga ttccgagcat tatgaacgtg ctgctggtgt gcctgatttt ttggctgatt 4080
          tttagcatta tgggcgtgaa cctgtttgcg ggcaaatttt atcattgcat taacaccacc 4140
          accggcgatc gctttgatat tgaagatgtg aacaaccata ccgattgcct gaaactgatt 4200
          gaacgcaacg aaaccgcgcg ctggaaaaac gtgaaagtga actttgataa cgtgggcttt 4260
          ggctatctga gcctgctgca ggtggcgacc tttaaaggct ggatggatat tatgtatgcg 4320
          gcggtggata gccgcaacgt ggaactgcag ccgaaatatg aagaaagcct gtatatgtat 4380
          ctgtattttg tgatttttat tatttttggc agctttttta ccctgaacct gtttattggc 4440
          gtgattattg ataactttaa ccagcagaaa aaaaaatttg gcggccagga tatttttatg 4500
          accgaagaac agaaaaaata ttataacgcg atgaaaaaac tgggcagcaa aaaaccgcag 4560
          aaaccgattc cgcgcccggg caacaaattt cagggcatgg tgtttgattt tgtgacccgc 4620
          caggtgtttg atattagcat tatgattctg atttgcctga acatggtgac catgatggtg 4680
          gaaaccgatg atcagagcga atatgtgacc accattctga gccgcattaa cctggtgttt 4740
          attgtgctgt ttaccggcga atgcgtgctg aaactgatta gcctgcgcca ttattatttt 4800
          accattggct ggaacatttt tgattttgtg gtggtgattc tgagcattgt gggcatgttt 4860
          ctggcggaac tgattgaaaa atattttgtg agcccgaccc tgtttcgcgt gattcgcctg 4920
          gcgcgcattg gccgcattct gcgcctgatt aaaggcgcga aaggcattcg caccctgctg 4980
          tttgcgctga tgatgagcct gccggcgctg tttaacattg gcctgctgct gtttctggtg 5040
          atgtttattt atgcgatttt tggcatgagc aactttgcgt atgtgaaacg cgaagtgggc 5100
          attgatgata tgtttaactt tgaaaccttt ggcaacagca tgatttgcct gtttcagatt 5160
          accaccagcg cgggctggga tggcctgctg gcgccgattc tgaacagcaa accgccggat 5220
          tgcgatccga acaaagtgaa cccgggcagc agcgtgaaag gcgattgcgg caacccgagc 5280
          gtgggcattt ttttttttgt gagctatatt attattagct ttctggtggt ggtgaacatg 5340
          tatattgcgg tgattctgga aaactttagc gtggcgaccg aagaaagcgc ggaaccgctg 5400
          agcgaagatg attttgaaat gttttatgaa gtgtgggaaa aatttgatcc ggatgcgacc 5460
          cagtttatgg aatttgaaaa actgagccag tttgcggcgg cgctggaacc gccgctgaac 5520
          ctgccgcagc cgaacaaact gcagctgatt gcgatggatc tgccgatggt gagcggcgat 5580
          cgcattcatt gcctggatat tctgtttgcg tttaccaaac gcgtgctggg cgaaagcggc 5640
          gaaatggatg cgctgcgcat tcagatggaa gaacgcttta tggcgagcaa cccgagcaaa 5700
          gtgagctatc agccgattac caccaccctg aaacgcaaac aggaagaagt gagcgcggtg 5760
          attattcagc gcgcgtatcg ccgccatctg ctgaaacgca ccgtgaaaca ggcgagcttt 5820
          acctataaca aaaacaaaat taaaggcggc gcgaacctgc tgattaaaga agatatgatt 5880
          attgatcgca ttaacgaaaa cagcattacc gaaaaaaccg atctgaccat gagcaccgcg 5940
          gcgtgcccgc cgagctatga tcgcgtgacc aaaccgattg tggaaaaaca tgaacaggaa 6000
          ggcaaagatg aaaaagcgaa aggcaaa 6027
           <![CDATA[ <210> 50]]>
           <![CDATA[ <211> 2009]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 50]]> Met Glu Gln Thr Val Leu Val Pro Pro Gly Pro Asp Ser Phe Asn Phe 1 5 10 15 Phe Thr Arg Glu Ser Leu Ala Ala Ile Glu Arg Arg Ile Ala Glu Glu 20 25 30 Lys Ala Lys Asn Pro Lys Pro Asp Lys Lys Asp Asp Asp Glu Asn Gly 35 40 45 Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Asn Leu Pro Phe Ile 50 55 60 Tyr Gly Asp Ile Pro Pro Glu Met Val Ser Glu Pro Leu Glu Asp Leu 65 70 75 80 Asp Pro Tyr Tyr Ile Asn Lys Lys Thr Phe Ile Val Leu Asn Lys Gly 85 90 95 Lys Ala Ile Phe Arg Phe Ser Ala Thr Ser Ala Leu Tyr Ile Leu Thr 100 105 110 Pro Phe Asn Pro Leu Arg Lys Ile Ala Ile Lys Ile Leu Val His Ser 115 120 125 Leu Phe Ser Met Leu Ile Met Cys Thr Ile Leu Thr Asn Cys Val Phe 130 135 140 Met Thr Met Ser Asn Pro Pro Asp Trp Thr Lys Asn Val Glu Tyr Thr 145 150 155 160 Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Ile Lys Ile Ile Ala Arg 165 170 175 Gly Phe Cys Leu Glu Asp Phe Thr Phe Leu Arg Asp Pro Trp Asn Trp 180 185 190 Leu Asp Phe Thr Val Ile Thr Phe Ala Tyr Val Thr Glu Phe Val Asp 195 200 205 Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu Arg Ala Leu 210 215 220 Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile Val Gly Ala Leu 225 230 235 240 Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile Leu Thr Val Phe 245 250 255 Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe Met Gly Asn 260 265 270 Leu Arg Asn Lys Cys Ile Gln Trp Pro Pro Thr Asn Ala Ser Leu Glu 275 280 285 Glu His Ser Ile Glu Lys Asn Ile Thr Val Asn Tyr Asn Gly Thr Leu 290 295 300 Ile Asn Glu Thr Val Phe Glu Phe Asp Trp Lys Ser Tyr Ile Gln Asp 305 310 315 320 Ser Arg Tyr His Tyr Phe Leu Glu Gly Phe Leu Asp Ala Leu Leu Cys 325 330 335 Gly Asn Ser Ser Asp Ala Gly Gln Cys Pro Glu Gly Tyr Met Cys Val 340 345 350 Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser Phe Asp Thr Phe 355 360 365 Ser Trp Ala Phe Leu Ser Leu Phe Arg Leu Met Thr Gln Asp Phe Trp 370 375 380 Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly Lys Thr Tyr Met 385 390 395 400 Ile Phe Phe Val Leu Val Ile Phe Leu Gly Ser Phe Tyr Leu Ile Asn 405 410 415 Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu Glu Gln Asn Gln Ala 420 425 430 Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu Phe Gln Gln Met Ile 435 440 445 Glu Gln Leu Lys Lys Gln Gln Glu Ala Ala Gln Gln Ala Ala Thr Ala 450 455 460 Thr Ala Ser Glu His Ser Arg Glu Pro Ser Ala Ala Gly Arg Leu Ser 465 470 475 480 Asp Ser Ser Ser Glu Ala Ser Lys Leu Ser Ser Lys Ser Ala Lys Glu 485 490 495 Arg Arg Asn Arg Arg Lys Lys Arg Lys Gln Lys Glu Gln Ser Gly Gly 500 505 510 Glu Glu Lys Asp Glu Asp Glu Phe Gln Lys Ser Glu Ser Glu Asp Ser 515 520 525 Ile Arg Arg Lys Gly Phe Arg Phe Ser Ile Glu Gly Asn Arg Leu Thr 530 535 540 Tyr Glu Lys Arg Tyr Ser Ser Pro His Gln Ser Leu Leu Ser Ile Arg 545 550 555 560 Gly Ser Leu Phe Ser Pro Arg Arg Asn Ser Arg Thr Ser Leu Phe Ser 565 570 575 Phe Arg Gly Arg Ala Lys Asp Val Gly Ser Glu Asn Asp Phe Ala Asp 580 585 590 Asp Glu His Ser Thr Phe Glu Asp Asn Glu Ser Arg Arg Asp Ser Leu 595 600 605 Phe Val Pro Arg Arg His Gly Glu Arg Arg Asn Ser Asn Leu Ser Gln 610 615 620 Thr Ser Arg Ser Ser Arg Met Leu Ala Val Phe Pro Ala Asn Gly Lys 625 630 635 640 Met His Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Val Gly Gly 645 650 655 Pro Ser Val Pro Thr Ser Pro Val Gly Gln Leu Leu Pro Glu Val Ile 660 665 670 Ile Asp Lys Pro Ala Thr Asp Asp Asn Gly Thr Thr Thr Glu Thr Glu 675 680 685 Met Arg Lys Arg Arg Ser Ser Ser Phe His Val Ser Met Asp Phe Leu 690 695 700 Glu Asp Pro Ser Gln Arg Gln Arg Ala Met Ser Ile Ala Ser Ile Leu 705 710 715 720 Thr Asn Thr Val Glu Glu Leu Glu Glu Ser Arg Gln Lys Cys Pro Pro 725 730 735 Cys Trp Tyr Lys Phe Ser Asn Ile Phe Leu Ile Trp Asp Cys Ser Pro 74 0 745 750 Tyr Trp Leu Lys Val Lys His Val Val Asn Leu Val Val Met Asp Pro 755 760 765 Phe Val Asp Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr Leu Phe 770 775 780 Met Ala Met Glu His Tyr Pro Met Thr Asp His Phe Asn Asn Val Leu 785 790 795 800 Thr Val Gly Asn Leu Val Phe Thr Gly Ile Phe Thr Ala Glu Met Phe 805 810 815 Leu Lys Ile Ile Ala Met Asp Pro Tyr Tyr Tyr Phe Gln Glu Gly Trp 820 825 830 Asn Ile Phe Asp Gly Phe Ile Val Thr Leu Ser Leu Val Glu Leu Gly 835 840 845 Leu Ala Asn Val Glu Gly Leu Ser Val Leu Arg Ser Phe Arg Leu Leu 850 855 860 Arg Val Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu Ile 865 870 875 880 Lys Ile Ile Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Th r Leu Val 885 890 895 Leu Ala Ile Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe 900 905 910 Gly Lys Ser Tyr Lys Asp Cys Val Cys Lys Ile Ala Ser Asp Cys Gln 915 920 925 Leu Pro Arg Trp His Met Asn Asp Phe Phe His Ser Phe Leu Ile Val 930 935 940 Phe Arg Val Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met 945 950 955 960 Glu Val Ala Gly Gln Ala Met Cys Leu Thr Val Phe Met Met Val Met 965 970 975 Val Ile Gly Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu 980 985 990 Ser Ser Phe Ser Ala Asp Asn Leu Ala Ala Thr Asp Asp Asp Asn Glu 995 1000 1005 Met Asn Asn Leu Gln Ile Ala Val Asp Arg Met His Lys Gly Val 1010 1015 1020 Ala Tyr Val Lys Arg Lys Ile Tyr Glu Phe Ile Gln Gln Ser Phe 1025 1030 1035 Ile Arg Lys Gln Lys Ile Leu Asp Glu Ile Lys Pro Le u Asp Asp 1040 1045 1050 Leu Asn Asn Lys Lys Asp Ser Cys Met Ser Asn His Thr Ala Glu 1055 1060 1065 Ile Gly Lys Asp Leu Asp Tyr Leu Lys Asp Val Asn Gly Thr Thr 1070 1075 1080 Ser Gly Ile Gly Thr Gly Ser Ser Val Glu Lys Tyr Ile Ile Asp 1085 1090 1095 Glu Ser Asp Tyr Met Ser Phe Ile Asn Asn Pro Ser Leu Thr Val 1100 1105 1110 Thr Val Pro Ile Ala Val Gly Glu Ser Asp Phe Glu Asn Leu Asn 1115 1120 1125 Thr Glu Asp Phe Ser Ser Glu Ser Asp Leu Glu Glu Ser Lys Glu 1130 1135 1140 Lys Leu Asn Glu Ser Ser Ser Ser Ser Glu Gly Ser Thr Val Asp 1145 1150 1155 Ile Gly Ala Pro Val Glu Glu Gln Pro Val Val Glu Pro Glu Glu 1160 1165 1170 Thr Leu Glu Pro Glu Ala Cys Phe Thr Glu Gly Cys Val Gln Arg 1175 1180 1185 Phe Lys Cys Cys Gln Ile Asn Val Glu Glu Glu Gly Arg Gly Lys Gln 1190 1195 1200 Trp Trp Asn Leu Arg Arg Thr Cys Phe Arg Ile Val Glu His Asn 1205 1210 1215 Trp Phe Glu Thr Phe Ile Val Phe Met Ile Leu Leu Ser Ser Gly 1220 1225 1230 Ala Leu Ala Phe Glu Asp Ile Tyr Ile Asp Gln Arg Lys Thr Ile 1235 1240 1245 Lys Thr Met Leu Glu Tyr Ala Asp Lys Val Phe Thr Tyr Ile Phe 1250 1255 1260 Ile Leu Glu Met Leu Leu Lys Trp Val Ala Tyr Gly Tyr Gln Thr 1265 1270 1275 Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu Ile Val Asp 1280 1285 1290 Val Ser Leu Val Ser Leu Thr Ala Asn Ala Leu Gly Tyr Ser Glu 1295 1300 1305 Leu Gly Ala Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg Pro 1310 1315 1320 Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn 1325 1330 1335 Ala Leu Leu Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val 1340 1345 1350 Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu 1355 1360 1365 Phe Ala Gly Lys Phe Tyr His Cys Ile Asn Thr Thr Thr Gly Asp 1370 1375 1380 Arg Phe Asp Ile Glu Asp Val Asn Asn His Thr Asp Cys Leu Lys 1385 1390 1395 Leu Ile Glu Arg Asn Glu Thr Ala Arg Trp Lys Asn Val Lys Val 1400 1405 1410 Asn Phe Asp Asn Val Gly Phe Gly Tyr Leu Ser Leu Leu Gln Val 1415 1420 1425 Ala Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp 1430 1435 1440 Ser Arg Asn Val Glu Leu G ln Pro Lys Tyr Glu Glu Ser Leu Tyr 1445 1450 1455 Met Tyr Leu Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe 1460 1465 1470 Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln 1475 1480 1485 Gln Lys Lys Lys Phe Gly Gly Gln Asp Ile Phe Met Thr Glu Glu 1490 1495 1500 Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Lys Leu Gly Ser Lys Lys 1505 1510 1515 Pro Gln Lys Pro Ile Pro Arg Pro Gly Asn Lys Phe Gln Gly Met 1520 1525 1530 Val Phe Asp Phe Val Thr Arg Gln Val Phe Asp Ile Ser Ile Met 1535 1540 1545 Ile Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Thr Asp 1550 1555 1560 Asp Gln Ser Glu Tyr Val Thr Thr Ile Leu Ser Arg Ile Asn Leu 1565 1570 1575 Val Phe Ile Val Leu Phe Thr Gly Glu Cys Val Leu Lys Leu Ile 1580 1585 1590 Ser Leu Arg His Tyr Tyr Phe Thr Ile Gly Trp Asn Ile Phe Asp 1595 1600 1605 Phe Val Val Val Ile Leu Ser Ile Val Gly Met Phe Leu Ala Glu 1610 1615 1620 Leu Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val Ile 1625 1630 1635 Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Ly s Gly Ala 1640 1645 1650 Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro 1655 1660 1665 Ala Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile 1670 1675 1680 Tyr Ala Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys Arg Glu 1685 1690 1695 Val Gly Ile Asp Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser 1700 1705 1710 Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly 1715 1720 1725 Leu Leu Ala Pro Ile Leu Asn Ser Lys Pro Pro Asp Cys Asp Pro 1730 1735 1740 Asn Lys Val Asn Pro Gly Ser Ser Val Lys Gly Asp Cys Gly Asn 1745 1750 1755 Pro Ser Val Gly Ile Phe Phe Phe Val Ser Tyr Ile Ile Ile Ser 1760 1765 1770 Phe Leu Val Val Val Asn Met Tyr Ile Ala Val Ile Leu Glu Asn 1775 1780 1785 Phe Ser Val Ala Thr Glu Glu Ser Ala Glu Pro Leu Ser Glu Asp 1790 1795 1800 Asp Phe Glu Met Phe Tyr Glu Val Trp Glu Lys Phe Asp Pro Asp 1805 1810 1815 Ala Thr Gln Phe Met Glu Phe Glu Lys Leu Ser Gln Phe Ala Ala 1820 1825 1830 Ala Leu Glu Pro Pro Leu Asn Leu Pro Gln Pro Asn Lys Leu Gln 1835 1840 1845 Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg Ile His 1850 1855 1860 Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly Glu 1865 1870 1875 Ser Gly Glu Met Asp Ala Leu Arg Ile Gln Met Glu Glu Arg Phe 1880 1885 1890 Met Ala Ser Asn Pro Ser Lys Val Ser Tyr Gln Pro Ile Thr Thr 1895 1900 1905 Thr Leu Lys Arg Lys Gln Glu Glu Val Ser Ala Val Ile Ile Gln 1910 1915 1920 Arg Ala Tyr Arg Arg His Leu Leu Lys Arg Thr Val Lys Gln Ala 1925 1930 1935 Ser Phe Thr Tyr Asn Lys Asn Lys Ile Lys Gly Gly Ala Asn Leu 1940 1945 1950 Leu Ile Lys Glu Asp Met Ile Ile Asp Arg Ile Asn Glu Asn Ser 1955 1960 1965 Ile Thr Glu Lys Thr Asp Leu Thr Met Ser Thr Ala Ala Cys Pro 1970 1975 1980 Pro Ser Tyr Asp Arg Val Thr Lys Pro Ile Val Glu Lys His Glu 1985 1990 1995 Gln Glu Gly Lys Asp Glu Lys Ala Lys Gly Lys 2000 2005 <![CDATA[ <210> 51]]>
           <![CDATA[ <211> 1470]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 51]]>
          atggatctgc tggtggatga actgtttgcg gatatgaacg cggatggcgc gagcccgccg 60
          ccgccgcgcc cggcgggcgg cccgaaaaac accccggcgg cgccgccgct gtatgcgacc 120
          ggccgcctga gccaggcgca gctgatgccg agcccgccga tgccggtgcc gccggcggcg 180
          ctgtttaacc gcctgctgga tgatctgggc tttagcgcgg gcccggcgct gtgcaccatg 240
          ctggatacct ggaacgaaga tctgtttagc gcgctgccga ccaacgcgga tctgtatcgc 300
          gaatgcaaat ttctgagcac cctgccgagc gatgtggtgg aatggggcga tgcgtatgtg 360
          ccggaacgca cccagattga tattcgcgcg catggcgatg tggcgtttcc gaccctgccg 420
          gcgacccgcg atggcctggg cctgtattat gaagcgctga gccgcttttt tcatgcggaa 480
          ctgcgcgcgc gcgaagaaag ctatcgcacc gtgctggcga acttttgcag cgcgctgtat 540
          cgctatctgc gcgcgagcgt gcgccagctg catcgccagg cgcatatgcg cggccgcgat 600
          cgcgatctgg gcgaaatgct gcgcgcgacc attgcggatc gctattatcg cgaaaccgcg 660
          cgcctggcgc gcgtgctgtt tctgcatctg tatctgtttc tgacccgcga aattctgtgg 720
          gcggcgtatg cggaacagat gatgcgcccg gatctgtttg attgcctgtg ctgcgatctg 780
          gaaagctggc gccagctggc gggcctgttt cagccgttta tgtttgtgaa cggcgcgctg 840
          accgtgcgcg gcgtgccgat tgaagcgcgc cgcctgcgcg aactgaacca tattcgcgaa 900
          catctgaacc tgccgctggt gcgcagcgcg gcgaccgaag aaccgggcgc gccgctgacc 960
          accccgccga ccctgcatgg caaccaggcg cgcgcgagcg gctattttat ggtgctgatt 1020
          cgcgcgaaac tggatagcta tagcagcttt accaccagcc cgagcgaagc ggtgatgcgc 1080
          gaacatgcgt atagccgcgc gcgcaccaaa aacaactatg gcagcaccat tgaaggcctg 1140
          ctggatctgc cggatgatga tgcgccggaa gaagcgggcc tggcggcgcc gcgcctgagc 1200
          tttctgccgg cgggccatac ccgccgcctg agcaccgcgc cgccgaccga tgtgagcctg 1260
          ggcgatgaac tgcatctgga tggcgaagat gtggcgatgg cgcatgcgga tgcgctggat 1320
          gattttgatc tggatatgct gggcgatggc gatagcccgg gcccgggctt taccccgcat 1380
          gatagcgcgc cgtatggcgc gctggatatg gcggattttg aatttgaaca gatgtttacc 1440
          gatgcgctgg gcattgatga atatggcggc 1470
           <![CDATA[ <210> 52]]>
           <![CDATA[ <211> 490]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 52]]>
          Met Asp Leu Leu Val Asp Glu Leu Phe Ala Asp Met Asn Ala Asp Gly
          1 5 10 15
          Ala Ser Pro Pro Pro Pro Arg Pro Ala Gly Gly Pro Lys Asn Thr Pro
                      20 25 30
          Ala Ala Pro Pro Leu Tyr Ala Thr Gly Arg Leu Ser Gln Ala Gln Leu
                  35 40 45
          Met Pro Ser Pro Pro Met Pro Val Pro Pro Ala Ala Leu Phe Asn Arg
              50 55 60
          Leu Leu Asp Asp Leu Gly Phe Ser Ala Gly Pro Ala Leu Cys Thr Met
          65 70 75 80
          Leu Asp Thr Trp Asn Glu Asp Leu Phe Ser Ala Leu Pro Thr Asn Ala
                          85 90 95
          Asp Leu Tyr Arg Glu Cys Lys Phe Leu Ser Thr Leu Pro Ser Asp Val
                      100 105 110
          Val Glu Trp Gly Asp Ala Tyr Val Pro Glu Arg Thr Gln Ile Asp Ile
                  115 120 125
          Arg Ala His Gly Asp Val Ala Phe Pro Thr Leu Pro Ala Thr Arg Asp
              130 135 140
          Gly Leu Gly Leu Tyr Tyr Glu Ala Leu Ser Arg Phe Phe His Ala Glu
          145 150 155 160
          Leu Arg Ala Arg Glu Glu Ser Tyr Arg Thr Val Leu Ala Asn Phe Cys
                          165 170 175
          Ser Ala Leu Tyr Arg Tyr Leu Arg Ala Ser Val Arg Gln Leu His Arg
                      180 185 190
          Gln Ala His Met Arg Gly Arg Asp Arg Asp Leu Gly Glu Met Leu Arg
                  195 200 205
          Ala Thr Ile Ala Asp Arg Tyr Tyr Arg Glu Thr Ala Arg Leu Ala Arg
              210 215 220
          Val Leu Phe Leu His Leu Tyr Leu Phe Leu Thr Arg Glu Ile Leu Trp
          225 230 235 240
          Ala Ala Tyr Ala Glu Gln Met Met Arg Pro Asp Leu Phe Asp Cys Leu
                          245 250 255
          Cys Cys Asp Leu Glu Ser Trp Arg Gln Leu Ala Gly Leu Phe Gln Pro
                      260 265 270
          Phe Met Phe Val Asn Gly Ala Leu Thr Val Arg Gly Val Pro Ile Glu
                  275 280 285
          Ala Arg Arg Leu Arg Glu Leu Asn His Ile Arg Glu His Leu Asn Leu
              290 295 300
          Pro Leu Val Arg Ser Ala Ala Thr Glu Glu Pro Gly Ala Pro Leu Thr
          305 310 315 320
          Thr Pro Pro Thr Leu His Gly Asn Gln Ala Arg Ala Ser Gly Tyr Phe
                          325 330 335
          Met Val Leu Ile Arg Ala Lys Leu Asp Ser Tyr Ser Ser Phe Thr Thr
                      340 345 350
          Ser Pro Ser Glu Ala Val Met Arg Glu His Ala Tyr Ser Arg Ala Arg
                  355 360 365
          Thr Lys Asn Asn Tyr Gly Ser Thr Ile Glu Gly Leu Leu Asp Leu Pro
              370 375 380
          Asp Asp Asp Ala Pro Glu Glu Ala Gly Leu Ala Ala Pro Arg Leu Ser
          385 390 395 400
          Phe Leu Pro Ala Gly His Thr Arg Arg Leu Ser Thr Ala Pro Pro Thr
                          405 410 415
          Asp Val Ser Leu Gly Asp Glu Leu His Leu Asp Gly Glu Asp Val Ala
                      420 425 430
          Met Ala His Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly
                  435 440 445
          Asp Gly Asp Ser Pro Gly Pro Gly Phe Thr Pro His Asp Ser Ala Pro
              450 455 460
          Tyr Gly Ala Leu Asp Met Ala Asp Phe Glu Phe Glu Gln Met Phe Thr
          465 470 475 480
          Asp Ala Leu Gly Ile Asp Glu Tyr Gly Gly
                          485 490
           <![CDATA[ <210> 53]]>
           <![CDATA[ <211> 2570]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 53]]>
          agcgcgcagg cgcggccgga ttccgggcag tgacgcgacg gcgggccgcg cggcgcattt 60
          ccgcctctgg cgaatggctc gtctgtagtg cacgccgcgg gcccagctgc gaccccggcc 120
          ccgccccccgg gaccccggcc atggacgaac tgttccccct catcttcccg gcagagccag 180
          cccaggcctc tggcccctat gtggagatca ttgagcagcc caagcagcgg ggcatgcgct 240
          tccgctacaa gtgcgagggg cgctccgcgg gcagcatccc aggcgagagg agcacagata 300
          ccaccaagac ccaccccacc atcaagatca atggctacac aggaccaggg acagtgcgca 360
          tctccctggt caccaaggac cctcctcacc ggcctcaccc ccacgagctt gtaggaaagg 420
          actgccggga tggcttctat gaggctgagc tctgcccgga ccgctgcatc cacagtttcc 480
          agaacctggg aatccagtgt gtgaagaagc gggacctgga gcaggctatc agtcagcgca 540
          tccagaccaa caacaacccc ttccaagaag agcagcgtgg ggactacgac ctgaatgctg 600
          tgcggctctg cttccaggtg acagtgcggg acccatcagg caggcccctc cgcctgccgc 660
          ctgtcctttc tcatcccatc tttgacaatc gtgcccccaa cactgccgag ctcaagatct 720
          gccgagtgaa ccgaaactct ggcagctgcc tcggtgggga tgagatcttc ctactgtgtg 780
          acaaggtgca gaaagaggac attgaggtgt atttcacggg accaggctgg gaggcccgag 840
          gctccttttc gcaagctgat gtgcaccgac aagtggccat tgtgttccgg acccctccct 900
          acgcagaccc cagcctgcag gctcctgtgc gtgtctccat gcagctgcgg cggccttccg 960
          accgggagct cagtgagccc atggaattcc agtacctgcc agatacagac gatcgtcacc 1020
          ggattgagga gaaacgtaaa aggacatatg agaccttcaa gagcatcatg aagaagagtc 1080
          ctttcagcgg acccaccgac ccccggcctc cacctcgacg cattgctgtg ccttcccgca 1140
          gctcagcttc tgtccccaag ccagcacccc agccctatcc ctttacgtca tccctgagca 1200
          ccatcaacta tgatgagttt cccaccatgg tgtttccttc tgggcagatc agccaggcct 1260
          cggccttggc cccggcccct ccccaagtcc tgccccaggc tccagcccct gcccctgctc 1320
          cagccatggt atcagctctg gcccaggccc cagcccctgt cccagtccta gccccaggcc 1380
          ctcctcaggc tgtggcccca cctgccccca agcccaccca ggctggggaa ggaacgctgt 1440
          cagaggccct gctgcagctg cagtttgatg atgaagacct gggggccttg cttggcaaca 1500
          gcacagaccc agctgtgttc acagacctgg catccgtcga caactccgag tttcagcagc 1560
          tgctgaacca gggcatacct gtggcccccc acacaactga gcccatgctg atggagtacc 1620
          ctgaggctat aactcgccta gtgacagggg cccagaggcc ccccgaccca gctcctgctc 1680
          cactggggc cccggggctc cccaatggcc tcctttcagg agatgaagac ttctcctcca 1740
          ttgcggacat ggacttctca gccctgctga gtcagatcag ctcctaaggg ggtgacgcct 1800
          gccctcccca gagcactggg ttgcagggga ttgaagccct ccaaaagcac ttacggattc 1860
          tggtggggtg tgttccaact gcccccaact ttgtggatgt cttccttgga ggggggagcc 1920
          atattttatt cttttattgt cagtatctgt atctctctct ctttttggag gtgcttaagc 1980
          agaagcatta acttctctgg aaagggggga gctggggaaa ctcaaacttt tcccctgtcc 2040
          tgatggtcag ctcccttctc tgtagggaac tctggggtcc cccatcccca tcctccagct 2100
          tctggtactc tcctagagac agaagcaggc tggaggtaag gcctttgagc ccacaaagcc 2160
          ttatcaagtg tcttccatca tggattcatt acagcttaat caaaataacg ccccagatac 2220
          cagcccctgt atggcactgg cattgtccct gtgcctaaca ccagcgtttg aggggctggc 2280
          cttcctgccc tacagaggtc tctgccggct ctttccttgc tcaaccatgg ctgaaggaaa 2340
          ccagtgcaac agcactggct ctctccagga tccagaaggg gtttggtctg ggacttcctt 2400
          gctctccctc ttctcaagtg ccttaatagt agggtaagtt gttaagagtg ggggagagca 2460
          ggctggcagc tctccagtca ggaggcatag tttttactga acaatcaaag cacttggact 2520
          cttgctcttt ctactctgaa ctaataaatc tgttgccaag ctggctagaa 2570
           <![CDATA[ <210> 54]]>
           <![CDATA[ <211> 548]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 54]]>
          Met Asp Glu Leu Phe Pro Leu Ile Phe Pro Ala Glu Pro Ala Gln Ala
          1 5 10 15
          Ser Gly Pro Tyr Val Glu Ile Ile Glu Gln Pro Lys Gln Arg Gly Met
                      20 25 30
          Arg Phe Arg Tyr Lys Cys Glu Gly Arg Ser Ala Gly Ser Ile Pro Gly
                  35 40 45
          Glu Arg Ser Thr Asp Thr Thr Lys Thr His Pro Thr Ile Lys Ile Asn
              50 55 60
          Gly Tyr Thr Gly Pro Gly Thr Val Arg Ile Ser Leu Val Thr Lys Asp
          65 70 75 80
          Pro Pro His Arg Pro His Pro His Glu Leu Val Gly Lys Asp Cys Arg
                          85 90 95
          Asp Gly Phe Tyr Glu Ala Glu Leu Cys Pro Asp Arg Cys Ile His Ser
                      100 105 110
          Phe Gln Asn Leu Gly Ile Gln Cys Val Lys Lys Arg Asp Leu Glu Gln
                  115 120 125
          Ala Ile Ser Gln Arg Ile Gln Thr Asn Asn Asn Pro Phe Gln Glu Glu
              130 135 140
          Gln Arg Gly Asp Tyr Asp Leu Asn Ala Val Arg Leu Cys Phe Gln Val
          145 150 155 160
          Thr Val Arg Asp Pro Ser Gly Arg Pro Leu Arg Leu Pro Pro Val Leu
                          165 170 175
          Ser His Pro Ile Phe Asp Asn Arg Ala Pro Asn Thr Ala Glu Leu Lys
                      180 185 190
          Ile Cys Arg Val Asn Arg Asn Ser Gly Ser Cys Leu Gly Gly Asp Glu
                  195 200 205
          Ile Phe Leu Leu Cys Asp Lys Val Gln Lys Glu Asp Ile Glu Val Tyr
              210 215 220
          Phe Thr Gly Pro Gly Trp Glu Ala Arg Gly Ser Phe Ser Gln Ala Asp
          225 230 235 240
          Val His Arg Gln Val Ala Ile Val Phe Arg Thr Pro Pro Tyr Ala Asp
                          245 250 255
          Pro Ser Leu Gln Ala Pro Val Arg Val Ser Met Gln Leu Arg Arg Pro
                      260 265 270
          Ser Asp Arg Glu Leu Ser Glu Pro Met Glu Phe Gln Tyr Leu Pro Asp
                  275 280 285
          Thr Asp Asp Arg His Arg Ile Glu Glu Lys Arg Lys Arg Thr Tyr Glu
              290 295 300
          Thr Phe Lys Ser Ile Met Lys Lys Ser Pro Phe Ser Gly Pro Thr Asp
          305 310 315 320
          Pro Arg Pro Pro Pro Arg Arg Ile Ala Val Pro Ser Arg Ser Ser Ala
                          325 330 335
          Ser Val Pro Lys Pro Ala Pro Gln Pro Tyr Pro Phe Thr Ser Ser Leu
                      340 345 350
          Ser Thr Ile Asn Tyr Asp Glu Phe Pro Thr Met Val Phe Pro Ser Gly
                  355 360 365
          Gln Ile Ser Gln Ala Ser Ala Leu Ala Pro Ala Pro Pro Gln Val Leu
              370 375 380
          Pro Gln Ala Pro Ala Pro Ala Pro Ala Pro Ala Met Val Ser Ala Leu
          385 390 395 400
          Ala Gln Ala Pro Ala Pro Val Pro Val Leu Ala Pro Gly Pro Pro Gln
                          405 410 415
          Ala Val Ala Pro Pro Ala Pro Lys Pro Thr Gln Ala Gly Glu Gly Thr
                      420 425 430
          Leu Ser Glu Ala Leu Leu Gln Leu Gln Phe Asp Asp Glu Asp Leu Gly
                  435 440 445
          Ala Leu Leu Gly Asn Ser Thr Asp Pro Ala Val Phe Thr Asp Leu Ala
              450 455 460
          Ser Val Asp Asn Ser Glu Phe Gln Gln Leu Leu Asn Gln Gly Ile Pro
          465 470 475 480
          Val Ala Pro His Thr Thr Glu Pro Met Leu Met Glu Tyr Pro Glu Ala
                          485 490 495
          Ile Thr Arg Leu Val Thr Gly Ala Gln Arg Pro Pro Asp Pro Ala Pro
                      500 505 510
          Ala Pro Leu Gly Ala Pro Gly Leu Pro Asn Gly Leu Leu Ser Gly Asp
                  515 520 525
          Glu Asp Phe Ser Ser Ile Ala Asp Met Asp Phe Ser Ala Leu Leu Ser
              530 535 540
          Gln Ile Ser Ser
          545
           <![CDATA[ <210> 55]]>
           <![CDATA[ <211> 1815]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 55]]>
          atgcgcccga aaaaagatgg cctggaagat tttctgcgcc tgaccccgga aattaaaaaa 60
          cagctgggca gcctggtgag cgattattgc aacgtgctga acaaagaatt taccgcgggc 120
          agcgtggaaa ttaccctgcg cagctataaa atttgcaaag cgtttattaa cgaagcgaaa 180
          gcgcatggcc gcgaatgggg cggcctgatg gcgaccctga acatttgcaa cttttgggcg 240
          attctgcgca acaaccgcgt gcgccgccgc gcggaaaacg cgggcaacga tgcgtgcagc 300
          attgcgtgcc cgattgtgat gcgctatgtg ctggatcatc tgattgtggt gaccgatcgc 360
          ttttttattc aggcgccgag caaccgcgtg atgattccgg cgaccattgg caccgcgatg 420
          tataaactgc tgaaacatag ccgcgtgcgc gcgtatacct atagcaaagt gctgggcgtg 480
          gatcgcgcgg cgattatggc gagcggcaaa caggtggtgg aacatctgaa ccgcatggaa 540
          aaagaaggcc tgctgagcag caaatttaaa gcgttttgca aatgggtgtt tacctatccg 600
          gtgctggaag aaatgtttca gaccatggtg agcagcaaaa ccggccatct gaccgatgat 660
          gtgaaagatg tgcgcgcgct gattaaaacc ctgccgcgcg cgagctatag cagccatgcg 720
          ggccagcgca gctatgtgag cggcgtgctg ccggcgtgcc tgctgagcac caaaagcaaa 780
          gcggtggaaa ccccgattct ggtgagcggc gcggatcgca tggatgaaga actgatgggc 840
          aacgatggcg gcgcgagcca taccgaagcg cgctatagcg aaagcggcca gtttcatgcg 900
          tttaccgatg aactggaaag cctgccgagc ccgaccatgc cgctgaaacc gggcgcgcag 960
          agcgcggatt gcggcgatag cagcagcagc agcagcgata gcggcaacag cgataccgaa 1020
          cagagcgaac gcgaagaagc gcgcgcggaa gcgccgcgcc tgcgcgcgcc gaaaagccgc 1080
          cgcaccagcc gcccgaaccg cggccagacc ccgtgcccga gcaacgcggc ggaaccggaa 1140
          cagccgtgga ttgcggcggt gcatcaggaa agcgatgaac gcccgatttt tccgcatccg 1200
          agcaaaccga cctttctgcc gccggtgaaa cgcaaaaaag gcctgcgcga tagccgcgaa 1260
          ggcatgtttc tgccgaaacc ggaagcgggc agcgcgatta gcgatgtgtt tgaaggccgc 1320
          gaagtgtgcc agccgaaacg cattcgcccg tttcatccgc cgggcagccc gtgggcgaac 1380
          cgcccgctgc cggcgagcct ggcgccgacc ccgaccggcc cggtgcatga accggtgggc 1440
          agcctgaccc cggcgccggt gccgcagccg ctggatccgg cgccggcggt gaccccggaa 1500
          gcgagccatc tgctggaaga tccggatgaa gaaaccagcc aggcggtgaa agcgctgcgc 1560
          gaaatggcgg ataccgtgat tccgcagaaa gaagaagcgg cgatttgcgg ccagatggat 1620
          ctgagccatc cgccgccgcg cggccatctg gatgaactga ccaccaccct ggaaagcatg 1680
          accgaagatc tgaacctgga tagcccgctg accccggaac tgaacgaaat tctggatacc 1740
          tttctgaacg atgaatgcct gctgcatgcg atgcatatta gcaccggcct gagcattttt 1800
          gataccagcc tgttt 1815
           <![CDATA[ <210> 56]]>
           <![CDATA[ <211> 605]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 56]]>
          Met Arg Pro Lys Lys Asp Gly Leu Glu Asp Phe Leu Arg Leu Thr Pro
          1 5 10 15
          Glu Ile Lys Lys Gln Leu Gly Ser Leu Val Ser Asp Tyr Cys Asn Val
                      20 25 30
          Leu Asn Lys Glu Phe Thr Ala Gly Ser Val Glu Ile Thr Leu Arg Ser
                  35 40 45
          Tyr Lys Ile Cys Lys Ala Phe Ile Asn Glu Ala Lys Ala His Gly Arg
              50 55 60
          Glu Trp Gly Gly Leu Met Ala Thr Leu Asn Ile Cys Asn Phe Trp Ala
          65 70 75 80
          Ile Leu Arg Asn Asn Arg Val Arg Arg Arg Ala Glu Asn Ala Gly Asn
                          85 90 95
          Asp Ala Cys Ser Ile Ala Cys Pro Ile Val Met Arg Tyr Val Leu Asp
                      100 105 110
          His Leu Ile Val Val Thr Asp Arg Phe Phe Ile Gln Ala Pro Ser Asn
                  115 120 125
          Arg Val Met Ile Pro Ala Thr Ile Gly Thr Ala Met Tyr Lys Leu Leu
              130 135 140
          Lys His Ser Arg Val Arg Ala Tyr Thr Tyr Ser Lys Val Leu Gly Val
          145 150 155 160
          Asp Arg Ala Ala Ile Met Ala Ser Gly Lys Gln Val Val Glu His Leu
                          165 170 175
          Asn Arg Met Glu Lys Glu Gly Leu Leu Ser Ser Lys Phe Lys Ala Phe
                      180 185 190
          Cys Lys Trp Val Phe Thr Tyr Pro Val Leu Glu Glu Met Phe Gln Thr
                  195 200 205
          Met Val Ser Ser Lys Thr Gly His Leu Thr Asp Asp Val Lys Asp Val
              210 215 220
          Arg Ala Leu Ile Lys Thr Leu Pro Arg Ala Ser Tyr Ser Ser His Ala
          225 230 235 240
          Gly Gln Arg Ser Tyr Val Ser Gly Val Leu Pro Ala Cys Leu Leu Ser
                          245 250 255
          Thr Lys Ser Lys Ala Val Glu Thr Pro Ile Leu Val Ser Gly Ala Asp
                      260 265 270
          Arg Met Asp Glu Glu Leu Met Gly Asn Asp Gly Gly Ala Ser His Thr
                  275 280 285
          Glu Ala Arg Tyr Ser Glu Ser Gly Gln Phe His Ala Phe Thr Asp Glu
              290 295 300
          Leu Glu Ser Leu Pro Ser Pro Thr Met Pro Leu Lys Pro Gly Ala Gln
          305 310 315 320
          Ser Ala Asp Cys Gly Asp Ser Ser Ser Ser Ser Ser Asp Ser Gly Asn
                          325 330 335
          Ser Asp Thr Glu Gln Ser Glu Arg Glu Glu Ala Arg Ala Glu Ala Pro
                      340 345 350
          Arg Leu Arg Ala Pro Lys Ser Arg Arg Thr Ser Arg Pro Asn Arg Gly
                  355 360 365
          Gln Thr Pro Cys Pro Ser Asn Ala Ala Glu Pro Glu Gln Pro Trp Ile
              370 375 380
          Ala Ala Val His Gln Glu Ser Asp Glu Arg Pro Ile Phe Pro His Pro
          385 390 395 400
          Ser Lys Pro Thr Phe Leu Pro Pro Val Lys Arg Lys Lys Gly Leu Arg
                          405 410 415
          Asp Ser Arg Glu Gly Met Phe Leu Pro Lys Pro Glu Ala Gly Ser Ala
                      420 425 430
          Ile Ser Asp Val Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg Ile
                  435 440 445
          Arg Pro Phe His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro Leu Pro
              450 455 460
          Ala Ser Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu Pro Val Gly
          465 470 475 480
          Ser Leu Thr Pro Ala Pro Val Pro Gln Pro Leu Asp Pro Ala Pro Ala
                          485 490 495
          Val Thr Pro Glu Ala Ser His Leu Leu Glu Asp Pro Asp Glu Glu Thr
                      500 505 510
          Ser Gln Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro
                  515 520 525
          Gln Lys Glu Glu Ala Ala Ile Cys Gly Gln Met Asp Leu Ser His Pro
              530 535 540
          Pro Pro Arg Gly His Leu Asp Glu Leu Thr Thr Thr Leu Glu Ser Met
          545 550 555 560
          Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu Asn Glu
                          565 570 575
          Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys Leu Leu His Ala Met His
                      580 585 590
          Ile Ser Thr Gly Leu Ser Ile Phe Asp Thr Ser Leu Phe
                  595 600 605
           <![CDATA[ <210> 57]]>
           <![CDATA[ <211> 172]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 57]]>
          Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Arg Gly
          1 5 10 15
          Asn Leu Val Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala
                      20 25 30
          Cys Asp Ile Cys Gly Lys Lys Phe Ala Leu Ser Phe Asn Leu Thr Arg
                  35 40 45
          His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile
              50 55 60
          Cys Met Arg Asn Phe Ser Arg Ser Asp Asn Leu Thr Arg His Ile Arg
          65 70 75 80
          Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Lys Lys
                          85 90 95
          Phe Ala Asp Arg Ser His Leu Ala Arg His Thr Lys Ile His Thr Gly
                      100 105 110
          Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln
                  115 120 125
          Lys Ala His Leu Thr Ala His Ile Arg Thr His Thr Gly Glu Lys Pro
              130 135 140
          Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Asp Asn Leu
          145 150 155 160
          Thr Arg His Thr Lys Ile His Leu Arg Gln Lys Asp
                          165 170
           <![CDATA[ <210> 58]]>
           <![CDATA[ <211> 516]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 58]]>
          cgaccattcc agtgtcgaat ctgcatgcgc aacttcagcc agcggggaaa cctggtgagg 60
          catatccgca cccacacggg agagaagcct tttgcctgcg atatttgtgg aaagaagttt 120
          gctctgagct tcaatctaac cagacacacc aagattcata ctgggtccca gaaaccgttc 180
          cagtgtagga tatgcatgag gaatttctct cggagtgaca acttaacgcg gcatataagg 240
          acgcacacag gtgaaaaacc atttgcatgc gacatctgtg gcaaaaagtt tgcggaccgg 300
          tctcaccttg cccgacacac aaaaatccat accggcagtc aaaagccctt tcaatgtcgc 360
          atttgcatgc gaaacttctc acagaaggcc catttgactg cccatattcg tactcatact 420
          ggcgagaaac ctttcgcttg cgatatatgt ggtcgtaagt ttgcacggtc ggacaacctc 480
          acacgccaca ctaagataca cctgcggcag aaggac 516
           <![CDATA[ <210> 59]]>
           <![CDATA[ <211> 172]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 59]]>
          Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Ser
          1 5 10 15
          Asn Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala
                      20 25 30
          Cys Asp Ile Cys Gly Lys Lys Phe Ala Asp Lys Arg Thr Leu Ile Arg
                  35 40 45
          His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile
              50 55 60
          Cys Met Arg Asn Phe Ser Gln Arg Gly Asn Leu Val Arg His Ile Arg
          65 70 75 80
          Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Lys Lys
                          85 90 95
          Phe Ala Leu Ser Phe Asn Leu Thr Arg His Thr Lys Ile His Thr Gly
                      100 105 110
          Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg
                  115 120 125
          Ser Asp Asn Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
              130 135 140
          Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser His Leu
          145 150 155 160
          Ala Arg His Thr Lys Ile His Leu Arg Gln Lys Asp
                          165 170
           <![CDATA[ <210> 60]]>
           <![CDATA[ <211> 516]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 60]]>
          cgaccattcc agtgtcgaat ctgcatgcgc aacttcagcc gaagttccaa cctgacacgg 60
          catatccgca cccacacggg agagaagcct tttgcctgcg atatttgtgg aaagaagttt 120
          gctgacaagc ggaccttaat ccgccacacc aagattcata ctgggtccca gaaaccgttc 180
          cagtgtagga tatgcatgag gaatttctct cagcggggaa atctagtgcg acatataagg 240
          acgcacacag gtgaaaaacc atttgcatgc gacatctgtg gcaaaaagtt tgcgctgagc 300
          ttcaacttga ctcgtcacac aaaaatccat accggcagtc aaaagccctt tcaatgtcgc 360
          atttgcatgc gaaacttctc acggagtgac aatcttacga gacatattcg tactcatact 420
          ggcgagaaac ctttcgcttg cgatatatgt ggtcgtaagt ttgcagaccg gagccactta 480
          gccaggcaca ctaagataca cctgcggcag aaggac 516
           <![CDATA[ <210> 61]]>
           <![CDATA[ <211> 172]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 61]]>
          Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Arg Ser
          1 5 10 15
          Ala Leu Ala Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala
                      20 25 30
          Cys Asp Ile Cys Gly Lys Lys Phe Ala Arg Ser Asp Asn Leu Thr Arg
                  35 40 45
          His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile
              50 55 60
          Cys Met Arg Asn Phe Ser Gln Ser Gly Asp Leu Thr Arg His Ile Arg
          65 70 75 80
          Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Lys Lys
                          85 90 95
          Phe Ala Val Arg Gln Thr Leu Lys Gln His Thr Lys Ile His Thr Gly
                      100 105 110
          Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Ala
                  115 120 125
          Ala Gly Asn Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
              130 135 140
          Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Asp Asn Leu
          145 150 155 160
          Thr Arg His Thr Lys Ile His Leu Arg Gln Lys Asp
                          165 170
           <![CDATA[ <210> 62]]>
           <![CDATA[ <211> 516]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 62]]>
          cgaccattcc agtgtcgaat ctgcatgcgc aacttcagcg accggagcgc gctggcacgg 60
          catatccgca cccacacggg agagaagcct tttgcctgcg atatttgtgg aaagaagttt 120
          gctcgaagtg acaacttaac gcgccacacc aagattcata ctgggtccca gaaaccgttc 180
          cagtgtagga tatgcatgag gaatttctct cagtcagggg acctcactcg tcatataagg 240
          acgcacacag gtgaaaaacc atttgcatgc gacatctgtg gcaaaaagtt tgcggtacga 300
          cagacgctta aacaacacac aaaaatccat accggcagtc aaaagccctt tcaatgtcgc 360
          atttgcatgc gaaacttctc agccgctggt aacttgacac gacatattcg tactcatact 420
          ggcgagaaac ctttcgcttg cgatatatgt ggtcgtaagt ttgcaagatc tgataatcta 480
          acgcgtcaca ctaagataca cctgcggcag aaggac 516
           <![CDATA[ <210> 63]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 63]]>
          Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Arg Gly Asn Leu
          1 5 10 15
          Val Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
                      20 25
           <![CDATA[ <210> 64]]>
           <![CDATA[ <211> 29]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 64]]>
          Phe Ala Cys Asp Ile Cys Gly Lys Lys Phe Ala Leu Ser Phe Asn Leu
          1 5 10 15
          Thr Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro
                      20 25
           <![CDATA[ <210> 65]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 65]]>
          Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp Asn Leu
          1 5 10 15
          Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
                      20 25
           <![CDATA[ <210> 66]]>
           <![CDATA[ <211> 29]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 66]]>
          Phe Ala Cys Asp Ile Cys Gly Lys Lys Phe Ala Asp Arg Ser His Leu
          1 5 10 15
          Ala Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro
                      20 25
           <![CDATA[ <210> 67]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 67]]>
          Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Lys Ala His Leu
          1 5 10 15
          Thr Ala His Ile Arg Thr His Thr Gly Glu Lys Pro
                      20 25
           <![CDATA[ <210> 68]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 68]]>
          Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Asp Asn Leu
          1 5 10 15
          Thr Arg His Thr Lys Ile His Leu Arg Gln Lys Asp
                      20 25
           <![CDATA[ <210> 69]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 69]]>
          Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Ser Asn Leu
          1 5 10 15
          Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
                      20 25
           <![CDATA[ <210> 70]]>
           <![CDATA[ <211> 29]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 70]]>
          Phe Ala Cys Asp Ile Cys Gly Lys Lys Phe Ala Asp Lys Arg Thr Leu
          1 5 10 15
          Ile Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro
                      20 25
           <![CDATA[ <210> 71]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 71]]>
          Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Arg Gly Asn Leu
          1 5 10 15
          Val Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
                      20 25
           <![CDATA[ <210> 72]]>
           <![CDATA[ <211> 29]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 72]]>
          Phe Ala Cys Asp Ile Cys Gly Lys Lys Phe Ala Leu Ser Phe Asn Leu
          1 5 10 15
          Thr Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro
                      20 25
           <![CDATA[ <210> 73]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 73]]>
          Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp Asn Leu
          1 5 10 15
          Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
                      20 25
           <![CDATA[ <210> 74]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 74]]>
          Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Asp Arg Ser His Leu
          1 5 10 15
          Ala Arg His Thr Lys Ile His Leu Arg Gln Lys Asp
                      20 25
           <![CDATA[ <210> 75]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 75]]>
          Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Arg Ser Ala Leu
          1 5 10 15
          Ala Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
                      20 25
           <![CDATA[ <210> 76]]>
           <![CDATA[ <211> 29]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 76]]>
          Phe Ala Cys Asp Ile Cys Gly Lys Lys Phe Ala Arg Ser Asp Asn Leu
          1 5 10 15
          Thr Arg His Thr Lys Ile His Thr Gly Ser Gln Lys Pro
                      20 25
           <![CDATA[ <210> 77]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 77]]>
          Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Gly Asp Leu
          1 5 10 15
          Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
                      20 25
           <![CDATA[ <210> 78]]>
           <![CDATA[ <211> 29]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 78]]>
          Phe Ala Cys Asp Ile Cys Gly Lys Lys Phe Ala Val Arg Gln Thr Leu
          1 5 10 15
          Lys Gln His Thr Lys Ile His Thr Gly Ser Gln Lys Pro
                      20 25
           <![CDATA[ <210> 79]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 79]]>
          Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Ala Ala Gly Asn Leu
          1 5 10 15
          Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro
                      20 25
           <![CDATA[ <210> 80]]>
           <![CDATA[ <211> 28]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 80]]>
          Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Asp Asn Leu
          1 5 10 15
          Thr Arg His Thr Lys Ile His Leu Arg Gln Lys Asp
                      20 25
           <![CDATA[ <210> 81]]>
           <![CDATA[ <211> 4104]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 81]]>
          atggacaaga agtactccat tgggctcgct atcggtacca acagcgtcgg ctgggccgtc 60
          attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
          cacagcataa agaagaacct cattggagcc ctcctgttcg actccgggga gacggccgaa 180
          gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
          tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
          ctggaggagt ccttttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
          aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
          aagctggtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcac 480
          atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
          gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga ggagaacccg 600
          atcaacgcat ccggcgttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
          cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
          cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
          gatgccaagc tgcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
          cagatcggcg accagtacgc agacctttttt ttggcggcaa agaacctgtc agacgccatt 900
          ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
          atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
          cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
          ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
          gaaaaaatgg acggcaccga ggagctgctg gtaaagctga acagagaaga tctgttgcgc 1200
          aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
          gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
          gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgctcg gggaaattcc 1380
          agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
          gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
          aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
          tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
          tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
          gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
          agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
          attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
          ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
          catctcttcg acgacaaagt catgaaacag ctcaagagac gccgatatac aggatggggg 1980
          cggctgtcaa gaaaactgat caatggcatc cgagacaagc agagtggaaa gacaatcctg 2040
          gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
          tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
          cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
          gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
          atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
          atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
          gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
          gacatgtacg tggatcagga actggacatc aaccggttgt ccgactacga cgtggatgct 2520
          atcgtgcccc aaagctttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
          gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
          aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
          actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
          cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
          accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
          aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
          taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
          tatcccaagc tggaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
          atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
          aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
          ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
          gcgacagtcc gcaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
          cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
          gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
          tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
          aaggaactgc tgggcatcac aatcatggag cgatccagct tcgagaaaaa ccccatcgac 3540
          tttctcgaag cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gctgcccaag 3600
          tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
          cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
          cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
          caacacaaac actaccttga tgagatcatc gagcaaataa gcgagttctc caaaagagtg 3840
          atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
          cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
          cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
          gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
          gacctctctc agctcggtgg agac 4104
           <![CDATA[ <210> 82]]>
           <![CDATA[ <211> 1368]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 82]]>
          Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
          1 5 10 15
          Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
                      20 25 30
          Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
                  35 40 45
          Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
              50 55 60
          Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
          65 70 75 80
          Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
                          85 90 95
          Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
                      100 105 110
          His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
                  115 120 125
          His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
              130 135 140
          Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
          145 150 155 160
          Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
                          165 170 175
          Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
                      180 185 190
          Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
                  195 200 205
          Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
              210 215 220
          Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
          225 230 235 240
          Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
                          245 250 255
          Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
                      260 265 270
          Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
                  275 280 285
          Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
              290 295 300
          Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
          305 310 315 320
          Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
                          325 330 335
          Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
                      340 345 350
          Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
                  355 360 365
          Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
              370 375 380
          Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
          385 390 395 400
          Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
                          405 410 415
          Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
                      420 425 430
          Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
                  435 440 445
          Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
              450 455 460
          Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
          465 470 475 480
          Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
                          485 490 495
          Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
                      500 505 510
          Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
                  515 520 525
          Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
              530 535 540
          Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
          545 550 555 560
          Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
                          565 570 575
          Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
                      580 585 590
          Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
                  595 600 605
          Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
              610 615 620
          Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
          625 630 635 640
          His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
                          645 650 655
          Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
                      660 665 670
          Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
                  675 680 685
          Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
              690 695 700
          Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
          705 710 715 720
          His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
                          725 730 735
          Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
                      740 745 750
          Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
                  755 760 765
          Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
              770 775 780
          Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
          785 790 795 800
          Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
                          805 810 815
          Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
                      820 825 830
          Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
                  835 840 845
          Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
              850 855 860
          Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
          865 870 875 880
          Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
                          885 890 895
          Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
                      900 905 910
          Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
                  915 920 925
          Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
              930 935 940
          Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
          945 950 955 960
          Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
                          965 970 975
          Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
                      980 985 990
          Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
                  995 1000 1005
          Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
              1010 1015 1020
          Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
              1025 1030 1035
          Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
              1040 1045 1050
          Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
              1055 1060 1065
          Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
              1070 1075 1080
          Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
              1085 1090 1095
          Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
              1100 1105 1110
          Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
              1115 1120 1125
          Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
              1130 1135 1140
          Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
              1145 1150 1155
          Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
              1160 1165 1170
          Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
              1175 1180 1185
          Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
              1190 1195 1200
          Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
              1205 1210 1215
          Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
              1220 1225 1230
          Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
              1235 1240 1245
          Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
              1250 1255 1260
          His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
              1265 1270 1275
          Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
              1280 1285 1290
          Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
              1295 1300 1305
          Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
              1310 1315 1320
          Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
              1325 1330 1335
          Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
              1340 1345 1350
          Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
              1355 1360 1365
           <![CDATA[ <210> 83]]>
           <![CDATA[ <211> 97]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 83]]>
          gaggtaccat agagtgaggc ggttttagag ctagaaatag caagttaaaa taaggctagt 60
          ccgttatcaa cttgaaaaag tggcaccgag tcggtgc 97
           <![CDATA[ <210> 84]]>
           <![CDATA[ <211> 97]]>
           <![CDATA[ <212> RNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 84]]>
          gagguaccau agagugaggc gguuuuagag cuagaaauag caaguuaaaa uaaggcuagu 60
          ccguuaucaa cuugaaaaag uggcaccgag ucggugc 97
           <![CDATA[ <210> 85]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 85]]>
          gaggtaccat agagtgaggc g 21
           <![CDATA[ <210> 86]]>
           <![CDATA[ <211> 21]]>
           <![CDATA[ <212> RNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 86]]>
          gagguaccau agagugaggc g 21
           <![CDATA[ <210> 87]]>
           <![CDATA[ <211> 99]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 87]]>
          accgaggcga ggatgaagcc gaggttttag agctagaaat agcaagttaa aataaggcta 60
          gtccgttatc aacttgaaaa agtggcaccg agtcggtgc 99
           <![CDATA[ <210> 88]]>
           <![CDATA[ <211> 99]]>
           <![CDATA[ <212> RNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 88]]>
          accgaggcga ggaugaagcc gagguuuuag agcuagaaau agcaaguuaa aauaaggcua 60
          guccguuauc aacuugaaaa aguggcaccg agucggugc 99
           <![CDATA[ <210> 89]]>
           <![CDATA[ <211> 23]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 89]]>
          accgaggcga ggatgaagcc gag 23
           <![CDATA[ <210> 90]]>
           <![CDATA[ <211> 23]]>
           <![CDATA[ <212> RNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 90]]>
          accgaggcga ggaugaagcc gag 23
           <![CDATA[ <210> 91]]>
           <![CDATA[ <211> 100]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 91]]>
          accgaagccg agaggatact gcaggtttta gagctagaaa tagcaagtta aaataaggct 60
          agtccgttat caacttgaaa aagtggcacc gagtcggtgc 100
           <![CDATA[ <210> 92]]>
           <![CDATA[ <211> 100]]>
           <![CDATA[ <212> RNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 92]]>
          accgaagccg agaggauacu gcagguuuua gagcuagaaa uagcaaguua aaauaaggcu 60
          aguccguuau caacuugaaa aaguggcacc gagucggugc 100
           <![CDATA[ <210> 93]]>
           <![CDATA[ <211> 24]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 93]]>
          accgaagccg agaggatact gcag 24
           <![CDATA[ <210> 94]]>
           <![CDATA[ <211> 24]]>
           <![CDATA[ <212> RNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 94]]>
          accgaagccg agaggauacu gcag 24
           <![CDATA[ <210> 95]]>
           <![CDATA[ <211> 1569]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 95]]>
          gacgcattgg acgattttga tctggatatg ctgggaagtg acgccctcga tgattttgac 60
          cttgacatgc ttggttcgga tgcccttgat gactttgacc tcgacatgct cggcagtgac 120
          gcccttgatg atttcgacct ggacatgctg attaactcta gaagttccgg atctccgaaa 180
          aagaaacgca aagttggtag ccagtacctg cccgacaccg acgaccggca ccggatcgag 240
          gaaaagcgga agcggaccta cgagacattc aagagcatca tgaagaagtc ccccttcagc 300
          ggccccaccg accctagacc tccacctaga agaatcgccg tgcccagcag atccagcgcc 360
          agcgtgccaa aacctgcccc ccagccttac cccttcacca gcagcctgag caccatcaac 420
          tacgacgagt tccctaccat ggtgttcccc agcggccaga tctctcaggc ctctgctctg 480
          gctccagccc ctcctcaggt gctgcctcag gctcctgctc ctgcaccagc tccagccatg 540
          gtgtctgcac tggctcaggc accagcaccc gtgcctgtgc tggctcctgg acctccacag 600
          gctgtggctc caccagcccc taaacctaca caggccggcg agggcacact gtctgaagct 660
          ctgctgcagc tgcagttcga cgacgaggat ctgggagccc tgctgggaaa cagcaccgat 720
          cctgccgtgt tcaccgacct ggccagcgtg gacaacagcg agttccagca gctgctgaac 780
          cagggcatcc ctgtggcccc tcacaccacc gagcccatgc tgatggaata ccccgaggcc 840
          atcacccggc tcgtgacagg cgctcagagg cctcctgatc cagctcctgc ccctctggga 900
          gcaccaggcc tgcctaatgg actgctgtct ggcgacgagg acttcagctc tatcgccgat 960
          atggatttct cagccttgct gggctctggc agcggcagcc gggattccag ggaagggatg 1020
          ttttttgccga agcctgaggc cggctccgct attagtgacg tgtttgaggg ccgcgaggtg 1080
          tgccagccaa aacgaatccg gccatttcat cctccaggaa gtccatgggc caaccgccca 1140
          ctccccgcca gcctcgcacc aacaccaacc ggtccagtac atgagccagt cgggtcactg 1200
          accccggcac cagtccctca gccactggat ccagcgcccg cagtgactcc cgaggccagt 1260
          cacctgttgg aggatcccga tgaagagacg agccaggctg tcaaagccct tcgggagatg 1320
          gccgatactg tgattcccca gaaggaagag gctgcaatct gtggccaaat ggacctttcc 1380
          catccgcccc caaggggcca tctggatgag ctgacaacca cacttgagtc catgaccgag 1440
          gatctgaacc tggactcacc cctgaccccg gaattgaacg agattctgga taccttcctg 1500
          aacgacgagt gcctcttgca tgccatgcat atcagcacag gactgtccat cttcgacaca 1560
          tctctgttt 1569
           <![CDATA[ <210> 96]]>
           <![CDATA[ <211> 523]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 96]]>
          Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu
          1 5 10 15
          Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe
                      20 25 30
          Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp
                  35 40 45
          Met Leu Ile Asn Ser Arg Ser Ser Gly Ser Pro Lys Lys Lys Arg Lys
              50 55 60
          Val Gly Ser Gln Tyr Leu Pro Asp Thr Asp Asp Arg His Arg Ile Glu
          65 70 75 80
          Glu Lys Arg Lys Arg Thr Tyr Glu Thr Phe Lys Ser Ile Met Lys Lys
                          85 90 95
          Ser Pro Phe Ser Gly Pro Thr Asp Pro Arg Pro Pro Pro Arg Arg Ile
                      100 105 110
          Ala Val Pro Ser Arg Ser Ser Ala Ser Val Pro Lys Pro Ala Pro Gln
                  115 120 125
          Pro Tyr Pro Phe Thr Ser Ser Leu Ser Thr Ile Asn Tyr Asp Glu Phe
              130 135 140
          Pro Thr Met Val Phe Pro Ser Gly Gln Ile Ser Gln Ala Ser Ala Leu
          145 150 155 160
          Ala Pro Ala Pro Pro Gln Val Leu Pro Gln Ala Pro Ala Pro Ala Pro
                          165 170 175
          Ala Pro Ala Met Val Ser Ala Leu Ala Gln Ala Pro Ala Pro Val Pro
                      180 185 190
          Val Leu Ala Pro Gly Pro Pro Gln Ala Val Ala Pro Pro Ala Pro Lys
                  195 200 205
          Pro Thr Gln Ala Gly Glu Gly Thr Leu Ser Glu Ala Leu Leu Gln Leu
              210 215 220
          Gln Phe Asp Asp Glu Asp Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp
          225 230 235 240
          Pro Ala Val Phe Thr Asp Leu Ala Ser Val Asp Asn Ser Glu Phe Gln
                          245 250 255
          Gln Leu Leu Asn Gln Gly Ile Pro Val Ala Pro His Thr Thr Glu Pro
                      260 265 270
          Met Leu Met Glu Tyr Pro Glu Ala Ile Thr Arg Leu Val Thr Gly Ala
                  275 280 285
          Gln Arg Pro Pro Asp Pro Ala Pro Ala Pro Leu Gly Ala Pro Gly Leu
              290 295 300
          Pro Asn Gly Leu Leu Ser Gly Asp Glu Asp Phe Ser Ser Ile Ala Asp
          305 310 315 320
          Met Asp Phe Ser Ala Leu Leu Gly Ser Gly Ser Gly Ser Arg Asp Ser
                          325 330 335
          Arg Glu Gly Met Phe Leu Pro Lys Pro Glu Ala Gly Ser Ala Ile Ser
                      340 345 350
          Asp Val Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg Ile Arg Pro
                  355 360 365
          Phe His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro Leu Pro Ala Ser
              370 375 380
          Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu Pro Val Gly Ser Leu
          385 390 395 400
          Thr Pro Ala Pro Val Pro Gln Pro Leu Asp Pro Ala Pro Ala Val Thr
                          405 410 415
          Pro Glu Ala Ser His Leu Leu Glu Asp Pro Asp Glu Glu Thr Ser Gln
                      420 425 430
          Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val Ile Pro Gln Lys
                  435 440 445
          Glu Glu Ala Ala Ile Cys Gly Gln Met Asp Leu Ser His Pro Pro Pro
              450 455 460
          Arg Gly His Leu Asp Glu Leu Thr Thr Thr Leu Glu Ser Met Thr Glu
          465 470 475 480
          Asp Leu Asn Leu Asp Ser Pro Leu Thr Pro Glu Leu Asn Glu Ile Leu
                          485 490 495
          Asp Thr Phe Leu Asn Asp Glu Cys Leu Leu His Ala Met His Ile Ser
                      500 505 510
          Thr Gly Leu Ser Ile Phe Asp Thr Ser Leu Phe
                  515 520
           <![CDATA[ <210> 97]]>
           <![CDATA[ <211> 1939]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 97]]> Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 74 0 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gl n Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Th r Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Pro Pro Lys Lys Lys Arg Lys Val Ala Ala Ala 1370 1375 1380 Ser Arg Tyr Pro Arg Gly Asp Ala Leu Asp Asp Phe Asp Leu Asp 1385 1390 1395 Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu 1400 1405 1410 Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser 1415 1420 1425 Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Ile Asn Ser Arg 1430 1435 1440 Ser Ser Gly Ser Pro Lys L ys Lys Arg Lys Val Gly Ser Gln Tyr 1445 1450 1455 Leu Pro Asp Thr Asp Asp Arg His Arg Ile Glu Glu Lys Arg Lys 1460 1465 1470 Arg Thr Tyr Glu Thr Phe Lys Ser Ile Met Lys Lys Ser Pro Phe 1475 1480 1485 Ser Gly Pro Thr Asp Pro Arg Pro Pro Pro Arg Arg Ile Ala Val 1490 1495 1500 Pro Ser Arg Ser Ser Ala Ser Val Pro Lys Pro Ala Pro Gln Pro 1505 1510 1515 Tyr Pro Phe Thr Ser Ser Leu Ser Thr Ile Asn Tyr Asp Glu Phe 1520 1525 1530 Pro Thr Met Val Phe Pro Ser Gly Gln Ile Ser Gln Ala Ser Ala 1535 1540 1545 Leu Ala Pro Ala Pro Pro Gln Val Leu Pro Gln Ala Pro Ala Pro 1550 1555 1560 Ala Pro Ala Pro Ala Met Val Ser Ala Leu Ala Gln Ala Pro Ala 1565 1570 1575 Pro Val Pro Val Leu Ala Pro Gly Pro Pro Gln Ala Val Ala Pro 1580 1585 1590 Pro Ala Pro Lys Pro Thr Gln Ala Gly Glu Gly Thr Leu Ser Glu 1595 1600 1605 Ala Leu Leu Gln Leu Gln Phe Asp Asp Glu Asp Leu Gly Ala Leu 1610 1615 1620 Leu Gly Asn Ser Thr Asp Pro Ala Val Phe Thr Asp Leu Ala Ser 1625 1630 1635 Val Asp Asn Ser Glu Phe Gln Gln Leu Leu Asn Gln Gl y Ile Pro 1640 1645 1650 Val Ala Pro His Thr Thr Glu Pro Met Leu Met Glu Tyr Pro Glu 1655 1660 1665 Ala Ile Thr Arg Leu Val Thr Gly Ala Gln Arg Pro Pro Asp Pro 1670 1675 1680 Ala Pro Ala Pro Leu Gly Ala Pro Gly Leu Pro Asn Gly Leu Leu 1685 1690 1695 Ser Gly Asp Glu Asp Phe Ser Ser Ile Ala Asp Met Asp Phe Ser 1700 1705 1710 Ala Leu Leu Gly Ser Gly Ser Gly Ser Arg Asp Ser Arg Glu Gly 1715 1720 1725 Met Phe Leu Pro Lys Pro Glu Ala Gly Ser Ala Ile Ser Asp Val 1730 1735 1740 Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg Ile Arg Pro Phe 1745 1750 1755 His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro Leu Pro Ala Ser 1760 1765 1770 Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu Pro Val Gly Ser 1775 1780 1785 Leu Thr Pro Ala Pro Val Pro Gln Pro Leu Asp Pro Ala Pro Ala 1790 1795 1800 Val Thr Pro Glu Ala Ser His Leu Leu Glu Asp Pro Asp Glu Glu 1805 1810 1815 Thr Ser Gln Ala Val Lys Ala Leu Arg Glu Met Ala Asp Thr Val 1820 1825 1830 Ile Pro Gln Lys Glu Glu Ala Ala Ile Cys Gly Gln Met Asp Leu 1835 1840 1845 Ser His Pro Pro Pro Arg Gly His Leu Asp Glu Leu Thr Thr Thr 1850 1855 1860 Leu Glu Ser Met Thr Glu Asp Leu Asn Leu Asp Ser Pro Leu Thr 1865 1870 1875 Pro Glu Leu Asn Glu Ile Leu Asp Thr Phe Leu Asn Asp Glu Cys 1880 1885 1890 Leu Leu His Ala Met His Ile Ser Thr Gly Leu Ser Ile Phe Asp 1895 1900 1905 Thr Ser Leu Phe Pro Lys Lys Lys Arg Lys Val Arg Ser Lys Arg 1910 1915 1920 Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Leu 1925 1930 1935 Asp <![CDATA[ <210> 98]]>
           <![CDATA[ <211> 112]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 98]]>
          gaaacaagct atttgctgat ttgtattagg taccatagag tgaggcgagg atgaagccga 60
          gaggatactg cagaggtctc tggtgcaatg tgtgtatgtg tgcgtttgtg tg 112
           <![CDATA[ <210> 99]]>
           <![CDATA[ <211> 71]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 99]]>
          gaaacaagct atttgctgat ttgtattagg taccatagag tgaggcgagg atgaagccga 60
          gaggatactg c 71
           <![CDATA[ <210> 100]]>
           <![CDATA[ <211> 112]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 100]]>
          gaaacaagct atttgctgat ttgtattagg taccatagag tgaggcgagg atgaagccga 60
          gaggatactg cagaggtctc tggtgcaatg tgtgtatgtg tgcgtttgtg tg 112
           <![CDATA[ <210> 101]]>
           <![CDATA[ <211> 108]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> misc_feature]]>
           <![CDATA[ <222> (52)..(52)]]>
           <![CDATA[ <223> n is a, c, g or t]]>
           <![CDATA[ <400> 101]]>
          gaaacaagct atttgctgat ttgtattagg taccatagag tgaggcgagg angaagccga 60
          gaggatactg cagaggtctc tggtgcaatg tgtgtatgtg tgcgtttg 108
           <![CDATA[ <210> 102]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 102]]>
          ttccagtgtc gaatctgcat gcgcaacttc agccagcggg gaaacctggt gaggcatatc 60
          cgcacccaca cgggagagaa gcct 84
           <![CDATA[ <210> 103]]>
           <![CDATA[ <211> 87]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 103]]>
          tttgcctgcg atatttgtgg aaagaagttt gctctgagct tcaatctaac cagacacacc 60
          aagattcata ctgggtccca gaaaccg 87
           <![CDATA[ <210> 104]]>
           <![CDATA[ <211> 85]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 104]]>
          ttccagtgta ggatatgcat gaggaatttc tctcggagtg acaacttaac gcggcatata 60
          aggacgcaca caggtgaaaa aacaa 85
           <![CDATA[ <210> 105]]>
           <![CDATA[ <211> 87]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 105]]>
          tttgcatgcg acatctgtgg caaaaagttt gcggaccggt ctcaccttgc ccgacacaca 60
          aaaatccata ccggcagtca aaagccc 87
           <![CDATA[ <210> 106]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 106]]>
          tttcaatgtc gcatttgcat gcgaaacttc tcacagaagg cccatttgac tgcccatatt 60
          cgtactcata ctggcgagaa acct 84
           <![CDATA[ <210> 107]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 107]]>
          ttcgcttgcg atatatgtgg tcgtaagttt gcacggtcgg acaacctcac acgccacact 60
          aagatacacc tgcggcagaa ggac 84
           <![CDATA[ <210> 108]]>
           <![CDATA[ <211> 85]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 108]]>
          ttccagtgtc gaatctgcat gcgcaacttc agcccgaatg tccaacctga cacggcatat 60
          ccgcacccac acgggagaga agcct 85
           <![CDATA[ <210> 109]]>
           <![CDATA[ <211> 87]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 109]]>
          tttgcctgcg atatttgtgg aaagaagttt gctgacaagc ggaccttaat ccgccacacc 60
          aagattcata ctgggtccca gaaaccg 87
           <![CDATA[ <210> 110]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 110]]>
          ttccagtgta ggatatgcat gaggaatttc tctcagcggg gaaatctagt gcgacatata 60
          aggacgcaca caggtgaaaa acca 84
           <![CDATA[ <210> 111]]>
           <![CDATA[ <211> 87]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 111]]>
          tttgcatgcg acatctgtgg caaaaagttt gcgctgagct tcaacttgac tcgtcacaca 60
          aaaatccata ccggcagtca aaagccc 87
           <![CDATA[ <210> 112]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 112]]>
          tttcaatgtc gcatttgcat gcgaaacttc tcacggagtg acaatcttac gagacatatt 60
          cgtactcata ctggcgagaa acct 84
           <![CDATA[ <210> 113]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 113]]>
          ttcgcttgcg atatatgtgg tcgtaagttt gcagaccgga gccacttagc caggcacact 60
          aagatacacc tgcggcagaa ggac 84
           <![CDATA[ <210> 114]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 114]]>
          ttccagtgtc gaatctgcat gcgcaacttc agcgaccgga gcgcgctggc acggcatatc 60
          cgcacccaca cgggagagaa gcct 84
           <![CDATA[ <210> 115]]>
           <![CDATA[ <211> 87]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 115]]>
          tttgcctgcg atatttgtgg aaagaagttt gctcgaagtg acaacttaac gcgccacacc 60
          aagattcata ctgggtccca gaaaccg 87
           <![CDATA[ <210> 116]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 116]]>
          ttccagtgta ggatatgcat gaggaatttc tctcagtcag gggacctcac tcgtcatata 60
          aggacgcaca caggtgaaaa acca 84
           <![CDATA[ <210> 117]]>
           <![CDATA[ <211> 87]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 117]]>
          tttgcatgcg acatctgtgg caaaaagttt gcggtacgac agacgcttaa acaacacaca 60
          aaaatccata ccggcagtca aaagccc 87
           <![CDATA[ <210> 118]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 118]]>
          tttcaatgtc gcatttgcat gcgaaacttc tcagccgctg gtaacttgac acgacatatt 60
          cgtactcata ctggcgagaa acct 84
           <![CDATA[ <210> 119]]>
           <![CDATA[ <211> 84]]>
           <![CDATA[ <212> DNA]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 119]]>
          ttcgcttgcg atatatgtgg tcgtaagttt gcaagatctg ataatctaac gcgtcacact 60
          aagatacacc tgcggcagaa ggac 84
           <![CDATA[ <210> 120]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthetic peptides]]>
           <![CDATA[ <400> 120]]>
          Thr Gly Glu Lys Pro
          1 5
           <![CDATA[ <210> 121]]>
           <![CDATA[ <211> 6]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthetic peptides]]>
           <![CDATA[ <400> 121]]>
          Thr Gly Ser Gln Lys Pro
          1 5
           <![CDATA[ <210> 122]]>
           <![CDATA[ <211> 98]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 122]]>
          Met His His Gln Gln Arg Met Ala Ala Leu Gly Thr Asp Lys Glu Leu
          1 5 10 15
          Ser Asp Leu Leu Asp Phe Ser Ala Met Phe Ser Pro Pro Val Ser Ser
                      20 25 30
          Gly Lys Asn Gly Pro Thr Ser Leu Ala Ser Gly His Phe Thr Gly Ser
                  35 40 45
          Asn Val Glu Asp Arg Ser Ser Ser Gly Ser Trp Gly Asn Gly Gly His
              50 55 60
          Pro Ser Pro Ser Arg Asn Tyr Gly Asp Gly Thr Pro Tyr Asp His Met
          65 70 75 80
          Thr Ser Arg Asp Leu Gly Ser His Asp Asn Leu Ser Pro Pro Phe Val
                          85 90 95
          Asn Ser
           <![CDATA[ <210> 123]]>
           <![CDATA[ <211> 72]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 123]]>
          Thr Asn Asn Ser Phe Ser Ser Asn Pro Ser Thr Pro Val Gly Ser Pro
          1 5 10 15
          Pro Ser Leu Ser Ala Gly Thr Ala Val Trp Ser Arg Asn Gly Gly Gln
                      20 25 30
          Ala Ser Ser Ser Pro Asn Tyr Glu Gly Pro Leu His Ser Leu Gln Ser
                  35 40 45
          Arg Ile Glu Asp Arg Leu Glu Arg Leu Asp Asp Ala Ile His Val Leu
              50 55 60
          Arg Asn His Ala Val Gly Pro Ser
          65 70
           <![CDATA[ <210> 124]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 124]]>
          Pro Leu Ser Glu Glu Glu Glu Glu Leu Glu Leu Asn Thr Gln Arg
          1 5 10
           <![CDATA[ <210> 125]]>
           <![CDATA[ <211> 13]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 125]]>
          Ser Val Ser Glu Asp Val Asp Leu Leu Leu Asn Gln Arg
          1 5 10
           <![CDATA[ <210> 126]]>
           <![CDATA[ <211> 14]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 126]]>
          His Leu Thr Glu Asp His Leu Asp Leu Asn Asn Ala Gln Arg
          1 5 10
           <![CDATA[ <210> 127]]>
           <![CDATA[ <211> 237]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 127]]>
          Asn Ser Val Ser Ala Ala Thr Leu Thr Pro Ser Ser Gln Ala Val Thr
          1 5 10 15
          Ile Ser Ser Ser Gly Ser Gln Glu Ser Gly Ser Gln Pro Val Thr Ser
                      20 25 30
          Gly Thr Thr Ile Ser Ser Ala Ser Leu Val Ser Ser Gln Ala Ser Ser
                  35 40 45
          Ser Ser Phe Phe Thr Asn Ala Asn Ser Tyr Ser Thr Thr Thr Thr Thr
              50 55 60
          Ser Asn Met Gly Ile Met Asn Phe Thr Thr Ser Gly Ser Ser Gly Thr
          65 70 75 80
          Asn Ser Gln Gly Gln Thr Pro Gln Arg Val Ser Gly Leu Gln Gly Ser
                          85 90 95
          Asp Ala Leu Asn Ile Gln Gln Asn Gln Thr Ser Gly Gly Ser Leu Gln
                      100 105 110
          Ala Gly Gln Gln Lys Glu Gly Glu Gln Asn Gln Gln Thr Gln Gln Gln
                  115 120 125
          Gln Ile Leu Ile Gln Pro Gln Leu Val Gln Gly Gly Gln Ala Leu Gln
              130 135 140
          Ala Leu Gln Ala Ala Pro Leu Ser Gly Gln Thr Phe Thr Thr Gln Ala
          145 150 155 160
          Ile Ser Gln Glu Thr Leu Gln Asn Leu Gln Leu Gln Ala Val Pro Asn
                          165 170 175
          Ser Gly Pro Ile Ile Ile Arg Thr Pro Thr Val Gly Pro Asn Gly Gln
                      180 185 190
          Val Ser Trp Gln Thr Leu Gln Leu Gln Asn Leu Gln Val Gln Asn Pro
                  195 200 205
          Gln Ala Gln Thr Ile Thr Leu Ala Pro Met Gln Gly Val Ser Leu Gly
              210 215 220
          Gln Thr Ser Ser Ser Asn Thr Thr Leu Thr Pro Ile Ala
          225 230 235
           <![CDATA[ <210> 128]]>
           <![CDATA[ <211> 94]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 128]]>
          Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln
          1 5 10 15
          Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu
                      20 25 30
          Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp
                  35 40 45
          Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro
              50 55 60
          Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro
          65 70 75 80
          Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser
                          85 90
           <![CDATA[ <210> 129]]>
           <![CDATA[ <211> 58]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 129]]>
          Ala Asp Ser Leu Leu Glu His Val Arg Glu Asp Phe Ser Gly Leu Leu
          1 5 10 15
          Pro Glu Glu Phe Ile Ser Leu Ser Pro Pro His Glu Ala Leu Asp Tyr
                      20 25 30
          His Phe Gly Leu Glu Glu Gly Glu Gly Ile Arg Asp Leu Phe Asp Cys
                  35 40 45
          Asp Phe Gly Asp Leu Thr Pro Leu Asp Phe
              50 55
           <![CDATA[ <210> 130]]>
           <![CDATA[ <211> 63]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 130]]>
          Met Glu Leu Leu Ser Pro Pro Leu Arg Asp Val Asp Leu Thr Ala Pro
          1 5 10 15
          Asp Gly Ser Leu Cys Ser Phe Ala Thr Thr Asp Asp Phe Tyr Asp Asp
                      20 25 30
          Pro Cys Phe Asp Ser Pro Asp Leu Arg Phe Phe Glu Asp Leu Asp Pro
                  35 40 45
          Arg Leu Met His Val Gly Ala Leu Leu Lys Pro Glu Glu His Ser
              50 55 60
           <![CDATA[ <210> 131]]>
           <![CDATA[ <211> 127]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 131]]>
          Leu Ala Ala Gln Ser Leu Val Pro Pro Pro Gly Leu Pro Gly Ser Ser
          1 5 10 15
          Thr Pro Gly Val Leu Pro Tyr Phe Pro Pro Gly Leu Pro Pro Pro Asp
                      20 25 30
          Ala Gly Gly Ala Pro Gln Ser Ser Met Ser Glu Ser Pro Asp Val Asn
                  35 40 45
          Leu Val Thr Gln Gln Leu Ser Lys Ser Gln Val Glu Asp Pro Leu Pro
              50 55 60
          Pro Val Phe Ser Gly Thr Pro Lys Gly Ser Gly Ala Gly Tyr Gly Val
          65 70 75 80
          Gly Phe Asp Leu Glu Glu Phe Leu Asn Gln Ser Phe Asp Met Gly Val
                          85 90 95
          Ala Asp Gly Pro Gln Asp Gly Gln Ala Asp Ser Ala Ser Leu Ser Ala
                      100 105 110
          Ser Leu Leu Ala Asp Trp Leu Glu Gly His Gly Met Asn Pro Ala
                  115 120 125
           <![CDATA[ <210> 132]]>
           <![CDATA[ <211> 102]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 132]]>
          Pro Glu Lys Pro Leu Phe Ser Ser Ala Ser Pro Gln Asp Ser Ser Pro
          1 5 10 15
          Arg Leu Ser Thr Phe Pro Gln His His His Pro Gly Ile Pro Gly Val
                      20 25 30
          Ala His Ser Val Ile Ser Thr Arg Thr Pro Pro Pro Pro Ser Pro Leu
                  35 40 45
          Pro Phe Pro Thr Gln Ala Ile Leu Pro Pro Ala Pro Ser Ser Tyr Phe
              50 55 60
          Ser His Pro Thr Ile Arg Tyr Pro Pro His Leu Asn Pro Gln Asp Thr
          65 70 75 80
          Leu Lys Asn Tyr Val Pro Ser Tyr Asp Pro Ser Ser Pro Gln Thr Ser
                          85 90 95
          Gln Ser Trp Tyr Leu Gly
                      100
           <![CDATA[ <210> 133]]>
           <![CDATA[ <211> 260]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 133]]>
          Gln Tyr Leu Pro Asp Thr Asp Asp Arg His Arg Ile Glu Glu Lys Arg
          1 5 10 15
          Lys Arg Thr Tyr Glu Thr Phe Lys Ser Ile Met Lys Lys Ser Pro Phe
                      20 25 30
          Ser Gly Pro Thr Asp Pro Arg Pro Pro Pro Arg Arg Ile Ala Val Pro
                  35 40 45
          Ser Arg Ser Ser Ala Ser Val Pro Lys Pro Ala Pro Gln Pro Tyr Pro
              50 55 60
          Phe Thr Ser Ser Leu Ser Thr Ile Asn Tyr Asp Glu Phe Pro Thr Met
          65 70 75 80
          Val Phe Pro Ser Gly Gln Ile Ser Gln Ala Ser Ala Leu Ala Pro Ala
                          85 90 95
          Pro Pro Gln Val Leu Pro Gln Ala Pro Ala Pro Ala Pro Ala Pro Ala
                      100 105 110
          Met Val Ser Ala Leu Ala Gln Ala Pro Ala Pro Val Pro Val Leu Ala
                  115 120 125
          Pro Gly Pro Pro Gln Ala Val Ala Pro Pro Ala Pro Lys Pro Thr Gln
              130 135 140
          Ala Gly Glu Gly Thr Leu Ser Glu Ala Leu Leu Gln Leu Gln Phe Asp
          145 150 155 160
          Asp Glu Asp Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp Pro Ala Val
                          165 170 175
          Phe Thr Asp Leu Ala Ser Val Asp Asn Ser Glu Phe Gln Gln Leu Leu
                      180 185 190
          Asn Gln Gly Ile Pro Val Ala Pro His Thr Thr Glu Pro Met Leu Met
                  195 200 205
          Glu Tyr Pro Glu Ala Ile Thr Arg Leu Val Thr Gly Ala Gln Arg Pro
              210 215 220
          Pro Asp Pro Ala Pro Ala Pro Leu Gly Ala Pro Gly Leu Pro Asn Gly
          225 230 235 240
          Leu Leu Ser Gly Asp Glu Asp Phe Ser Ser Ile Ala Asp Met Asp Phe
                          245 250 255
          Ser Ala Leu Leu
                      260
           <![CDATA[ <210> 134]]>
           <![CDATA[ <211> 124]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 134]]>
          Gly Phe Ser Val Asp Thr Ser Ala Leu Leu Asp Leu Phe Ser Pro Ser
          1 5 10 15
          Val Thr Val Pro Asp Met Ser Leu Pro Asp Leu Asp Ser Ser Leu Ala
                      20 25 30
          Ser Ile Gln Glu Leu Leu Ser Pro Gln Glu Pro Pro Arg Pro Pro Glu
                  35 40 45
          Ala Glu Asn Ser Ser Pro Asp Ser Gly Lys Gln Leu Val His Tyr Thr
              50 55 60
          Ala Gln Pro Leu Phe Leu Leu Asp Pro Gly Ser Val Asp Thr Gly Ser
          65 70 75 80
          Asn Asp Leu Pro Val Leu Phe Glu Leu Gly Glu Gly Ser Tyr Phe Ser
                          85 90 95
          Glu Gly Asp Gly Phe Ala Glu Asp Pro Thr Ile Ser Leu Leu Thr Gly
                      100 105 110
          Ser Glu Pro Pro Lys Ala Lys Asp Pro Thr Val Ser
                  115 120
           <![CDATA[ <210> 135]]>
           <![CDATA[ <211> 8]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 135]]>
          Pro Lys Lys Lys Arg Lys Val Glu
          1 5
           <![CDATA[ <210> 136]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 136]]>
          Pro Ala Ala Lys Arg Val Lys Leu Asp
          1 5
           <![CDATA[ <210> 137]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 137]]>
          Pro Ala Ala Lys Lys Lys Lys Lys Leu Asp
          1 5
           <![CDATA[ <210> 138]]>
           <![CDATA[ <211> 18]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 138]]>
          Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
          1 5 10 15
          Leu Asp
           <![CDATA[ <210> 139]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 139]]>
          Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Thr Pro Lys Lys Lys
          1 5 10 15
          Arg Lys Val Glu
                      20
           <![CDATA[ <210> 140]]>
           <![CDATA[ <211> 23]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Synthesis]]>
           <![CDATA[ <400> 140]]>
          Pro Arg Arg Arg Pro Leu His Ser Ser Ala Met Glu Val Gln Thr Lys
          1 5 10 15
          Lys Val Arg Lys Val Pro Pro
                      20
          
      

Claims (58)

An isolated nucleic acid comprising a transgene configured to express at least one DNA binding domain fused to at least one transcriptional regulatory domain, wherein the DNA binding domain binds to a target gene or a regulatory region of a target gene, wherein the target gene encodes a potential gated sodium channel, and wherein the at least one transcriptional regulatory domain comprises TCF4 transactivator, MEF2A transactivator, MEF2C transactivator, MEF2D transactivator, Sp1 glutamate-rich transactivator, p53 transactivator domain, E2F1 A transactivator, a MyoD transactivator, a MAPK7 transactivator domain, a NF1B proline-rich transactivator, or a RelA transactivator, or any combination thereof. The isolated nucleic acid of claim 1, wherein the transgene additionally comprises a nuclear localization sequence. An isolated nucleic acid comprising a transgene configured to express at least one DNA binding domain fused to at least one transcriptional regulatory domain, wherein the DNA binding domain binds to a target gene or a regulatory region of a target gene, wherein the target gene encodes a potential gated sodium channels, and wherein the transgene comprises a nuclear localization sequence. The isolated nucleic acid of claim 3, wherein the at least one transcriptional regulatory domain comprises VPR transactivator, Rta transactivator, p65 transactivator, Hsf1 transactivator, TCF4 transactivator, MEF2A transactivator, MEF2C transactivator MEF2D transactivator, Sp1 glutamate-rich transactivator, p53 transactivator domain, E2F1 transactivator, MyoD transactivator, MAPK7 transactivator domain, NF1B proline-rich transactivator, or RelA transactivator sub or any combination thereof. The isolated nucleic acid of any one of claims 2 to 4, wherein the nuclear localization sequence comprises any one of SEQ ID NOs: 135 to 140. The isolated nucleic acid of any one of claims 2 to 4, wherein the nuclear localization sequence comprises any combination of SEQ ID NOs: 135 to 140. The isolated nucleic acid of any one of claims 1 to 6, wherein the transgenic line is flanked by inverted terminal repeats (ITRs) derived from adeno-associated virus (AAV). The isolated nucleic acid of any one of claims 1 to 7, wherein the transcriptional regulatory domain upregulates the expression of the target gene. The isolated nucleic acid of any one of claims 1 to 8, wherein the at least one DNA binding domain binds to a non-translated region of the target gene. The isolated nucleic acid of claim 9, wherein the non-translated region is an enhancer, promoter, intron and/or repressor. The isolated nucleic acid of any one of claims 1 to 10, wherein the DNA binding domain binds between 2 and 2000 bp upstream or between 2 and 2000 bp downstream of the regulatory region of the target gene. The isolated nucleic acid of any one of claims 1 to 11, wherein the at least one DNA binding domain encodes a zinc finger protein (ZFP), a transcription-activator like effector (TALE), dCas proteins (eg, dCas9 or dCas12a) and/or homeodomains. The isolated nucleic acid of any one of claims 1 to 12, wherein the at least one DNA binding domain binds to the nucleic acid sequence set forth in any one of SEQ ID NOs: 5 to 7. The isolated nucleic acid of any one of claims 1 to 13, wherein the at least one DNA binding domain binds to at least 2 (eg, at least 3, 4, 5, 6, 7) of the nucleic acid sequence set forth in SEQ ID NO: 3 , 8, 9, 10 or more) consecutive nucleotides. The isolated nucleic acid of any one of claims 1 to 14, wherein the at least one DNA binding domain is a zinc finger protein comprising a recognition helix consisting of SEQ ID NOs: 11 to 16, 23 to 28, or 35 to Nucleic acid encoding of the sequences set forth in any of 40. The isolated nucleic acid of claim 15, wherein: (i) the at least one DNA binding domain is a zinc finger protein comprising a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 11, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 12, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 12, The recognition helix encoded by the nucleic acid comprising SEQ ID NO: 13, the recognition helix encoded by the nucleic acid comprising SEQ ID NO: 14, the recognition helix encoded by the nucleic acid comprising SEQ ID NO: 15 and/or the nucleic acid comprising SEQ ID NO: 16 The identification helix of the code; (ii) the at least one DNA binding domain is a zinc finger protein comprising a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 23, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 24, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 24, The recognition helix encoded by the nucleic acid comprising SEQ ID NO:25, the recognition helix encoded by the nucleic acid comprising SEQ ID NO:26, the recognition helix encoded by the nucleic acid comprising SEQ ID NO:27 and/or the nucleic acid comprising SEQ ID NO:28 an encoded identification helix; or (iii) the at least one DNA binding domain is a zinc finger protein comprising a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 35, a recognition helix encoded by a nucleic acid comprising SEQ ID NO: 36, The recognition helix encoded by the nucleic acid comprising SEQ ID NO:37, the recognition helix encoded by the nucleic acid comprising SEQ ID NO:38, the recognition helix encoded by the nucleic acid comprising SEQ ID NO:39 and/or the nucleic acid comprising SEQ ID NO:40 The identification helix of the code. The isolated nucleic acid of any one of claims 1 to 14, wherein the at least one DNA binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17 to 22, 29 to 34, or 41 to 46 the zinc finger protein. The isolated nucleic acid of claim 17, wherein: (i) the at least one DNA binding domain is a zinc finger protein comprising a recognition helix comprising SEQ ID NO: 17, a recognition helix comprising SEQ ID NO: 18, a recognition helix comprising SEQ ID NO: 19, comprising the recognition helix of SEQ ID NO: 20, the recognition helix comprising SEQ ID NO: 21 and/or the recognition helix comprising SEQ ID NO: 22; (ii) the at least one DNA binding domain is a zinc finger protein comprising a recognition helix comprising SEQ ID NO: 29, a recognition helix comprising SEQ ID NO: 30, a recognition helix comprising SEQ ID NO: 31, comprising The recognition helix of SEQ ID NO:32, the recognition helix comprising SEQ ID NO:33 and/or the recognition helix comprising SEQ ID NO:34; or (iii) the at least one DNA binding domain is a zinc finger protein comprising a recognition helix comprising SEQ ID NO: 41, a recognition helix comprising SEQ ID NO: 42, a recognition helix comprising SEQ ID NO: 43, comprising The recognition helix of SEQ ID NO:44, the recognition helix comprising SEQ ID NO:45 and/or the recognition helix comprising SEQ ID NO:46. The isolated nucleic acid of any one of claims 1 to 18, wherein the at least one DNA binding domain is a dCas protein, optionally a dCas9 protein, and optionally wherein the isolated nucleic acid additionally comprises at least one guide nucleic acid. The isolated nucleic acid of claim 19, wherein the guide nucleic acid comprises a spacer sequence targeting SCN1A . The isolated nucleic acid of claim 19 or 20, wherein the guide nucleic acid comprises a spacer sequence having the nucleotide sequence of any one of SEQ ID NOs: 85, 86, 89, 90, 93 or 94. The isolated nucleic acid of any one of claims 19 to 21, wherein the guide nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 83 to 94. The isolated nucleic acid of any one of claims 1 to 22, wherein the at least one transcriptional regulatory domain is encoded by the amino acid sequence set forth in any one of SEQ ID NOs: 122 to 134. The isolated nucleic acid of any one of claims 1 to 23, wherein the nucleic acid comprises AAV2 ITR. The isolated nucleic acid of claim 24, wherein the ITR is ΔTR and/or mTR. The isolated nucleic acid of any one of claims 1 to 25, wherein the transgenic line is operably linked to a promoter. The isolated nucleic acid of claim 26, wherein the promoter is a tissue-specific promoter, optionally wherein the promoter is a neuronal promoter, such as SST, NPY, Phosphate-activated glutaminase ; PAG), Vesicular glutamate transporter-1 (VGLUT1), Glutamate decarboxylase 65 and 57 (GAD65, GAD67), Synaptophysin I, a-CamKII, Dock10, Prox1 , microalbumin (PV), somatostatin (Somatostatin; SST), cholecystokinin (Cholecystokinin; CCK), calcium binding protein (Calretinin; CR) or neuropeptide Y (Neuropeptide Y; NPY). The isolated nucleic acid of any one of claims 1 to 27, wherein the at least one DNA binding domain is fused to the at least one transcriptional regulatory domain via a linker domain. The isolated nucleic acid of claim 28, wherein the linker domain is selectively: (i) a flexible linker, optionally consisting of glycine, or (ii) Cleavable linkers. The isolated nucleic acid of any one of claims 1 to 29, wherein the transgene encodes 1 DNA binding domain, 2 DNA binding domains, 3 DNA binding domains, 4 DNA binding domains, 5 DNA binding domains, 6 DNA binding domains DNA binding domains, 7 DNA binding domains, 8 DNA binding domains, 9 DNA binding domains, or 10 DNA binding domains. The isolated nucleic acid of any one of claims 1 to 30, wherein the transgene encodes 1 transcriptional regulatory domain, 2 transcriptional regulatory domains, 3 transcriptional regulatory domains, 4 transcriptional regulatory domains, 5 transcriptional regulatory domains, 6 1 transcriptional regulatory domain, 7 transcriptional regulatory domains, 8 transcriptional regulatory domains, 9 transcriptional regulatory domains, or 10 transcriptional regulatory domains. A recombinant AAV (rAAV) comprising: (i) the isolated nucleic acid of any one of claims 1 to 31, (ii) at least one capsid protein. The rAAV of claim 32, wherein the AAV capsid protein serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10 or AAV.PHPB . A method of increasing expression of a target gene, the method comprising administering to a cell or individual comprising the target gene an isolated nucleic acid as claimed in any one of claims 1 to 31 or an rAAV as claimed in claim 32 or 33. The method of claim 34, wherein the systemic target gene is haploinsufficient. The method of claim 34 or 35, wherein the target gene is SCN1A. The method of any one of claims 34 to 36, wherein the cell line neurons are selectively GABAergic neurons. The method of any one of claims 34 to 37, wherein administration of the isolated nucleic acid of any one of claims 1 to 31 or the rAAV of claim 32 or 33 results in the expression of the target gene relative to the expression of the target gene prior to administration The expression of the transgene in the individual is increased by at least 2-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold times. A method of treating Dravet syndrome in an individual comprising administering an isolated nucleic acid as claimed in any one of claims 1 to 31 or an rAAV as claimed in claim 32 or 33 to an individual expressing a target gene. The method of claim 39, wherein the expression of the target gene is reduced in the individual compared to a normal individual. The method of claim 39 or 40, wherein the system or suspected line target gene exhibits haploinsufficiency relative to normal individuals. The method of any one of claims 39 to 41, wherein the individual has or is suspected of having a disorder caused by a haploinsufficiency expression of the target gene. The method of any one of claims 39 to 42, wherein the target gene is SCN1A. The method of any one of claims 39 to 43, wherein the isolated nucleic acid or the rAAV is administered by intravenous injection, intramuscular injection, inhalation, subcutaneous injection and/or intracranial injection. The method of any one of claims 39 to 44, wherein administration of the isolated nucleic acid or the rAAV results in at least a 2-fold, at least 10-fold increase in target gene expression relative to the expression of the transgene in the individual prior to administration, At least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times. A composition comprising an isolated nucleic acid as claimed in any one of claims 1 to 31 or an rAAV as claimed in claim 32 or 33. The composition of claim 46, further comprising a pharmaceutically acceptable carrier. A kit comprising: A container containing an isolated nucleic acid as claimed in any one of claims 1 to 31 or an rAAV as claimed in claim 32 or 33. The kit of claim 48, wherein the kit additionally comprises a container containing a pharmaceutically acceptable carrier. The kit of claim 48 or 49, wherein the isolated nucleic acid of rAAV and the pharmaceutically acceptable carrier are contained in the same container. The kit of any one of claims 48 to 50, wherein the container is a syringe. A host cell comprising the isolated nucleic acid of any one of claims 1 to 31 or the rAAV of claim 32 or 33. The host cell of claim 52, wherein the host cell is a eukaryotic cell. The host cell of claim 52, wherein the host cell is a mammalian cell, optionally a human cell, optionally a neuron, optionally a GABAergic neuron. A method of identifying a GABAergic promoter comprising: (i) Bioinformatics mining of neuronal promoters to select candidate enhancer elements; (ii) each candidate enhancer element is individually fused to the minimal promoter linked to the transgene, thereby generating a pool of candidate promoters linked to the transgene; (iii) delivering each transgene of the pool to an individual; and (iv) Assess the performance of each transgene in the individual GABA neurons relative to non-target tissues. The method of claim 55, wherein each transgenic line of the pool in (iii) is delivered simultaneously to the same individual. The method of claim 55 or 56, wherein each transgene comprises a unique barcode. The method of any one of claims 55 to 57, wherein the neuronal promoter is a SCN1A, GAD1 or GAD2 promoter.

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